Heterologous Expression of Termite Cellulases Yeast

ABSTRACT

The present invention provides for heterologous expression of termite and termite-associated symbiont cellulases. The cellulases can, for example, be codon-optimized and expressed in yeast host cells, such as the yeast  Saccharomyces cerevisiae . The cellulases can also be co-expressed in host cells with other cellulases. The expression in such host cells of the termite and termite-associated symbiont cellulases, and variants and combinations thereof, result in yeast with improved cellulosic activity. Thus, such genes and expression systems are useful for efficient and cost-effective consolidated bioprocessing systems.

BACKGROUND OF THE INVENTION

Lignocellulosic biomass is widely recognized as a promising source ofraw material for production of renewable fuels and chemicals. Theprimary obstacle impeding the more widespread production of energy frombiomass feedstocks is the general absence of low-cost technology forovercoming the recalcitrance of these materials to conversion intouseful fuels. Lignocellulosic biomass contains carbohydrate fractions(e.g., cellulose and hemicellulose) that can be converted into ethanol.In order to convert these fractions, the cellulose and hemicellulosemust ultimately be converted or hydrolyzed into monosaccharides; it isthe hydrolysis that has historically proven to be problematic.

Biologically mediated processes are promising options for energyconversion, in particular for the conversion of lignocellulosic biomassinto fuels. Biomass processing schemes involving enzymatic or microbialhydrolysis commonly involve four biologically mediated transformations:(1) the production of saccharolytic enzymes (cellulases andhemicellulases); (2) the hydrolysis of carbohydrate components presentin pretreated biomass to sugars; (3) the fermentation of hexose sugars(e.g., glucose, mannose, and galactose); and (4) the fermentation ofpentose sugars (e.g., xylose and arabinose). These four transformationsoccur in a single step in a process configuration called consolidatedbioprocessing (CBP), which is distinguished from other less integratedconfigurations in that it does not involve a dedicated process step forcellulase and/or hemicellulase production.

CBP offers the potential for lower cost and higher efficiency thanprocesses featuring dedicated cellulase production. The benefits resultin part from avoiding capital costs associated for example, withsubstrates, raw materials and utilities required for cellulaseproduction. In addition, several factors support the realization ofhigher rates of hydrolysis, and hence reduced reactor volume and capitalinvestment using CBP, including enzyme-microbe synergy and the use ofthermophilic organisms and/or complexed cellulase systems. Moreover,cellulose-adherent cellulolytic microorganisms are likely to competesuccessfully for products of cellulose hydrolysis with non-adheredmicrobes, e.g., contaminants, which could increase the stability ofindustrial processes based on microbial cellulose utilization. Progressin developing CBP-enabling microorganisms is being made through twostrategies: engineering naturally occurring cellulolytic microorganismsto improve product-related properties, such as yield and titer; andengineering non-cellulolytic organisms that exhibit high product yieldsand titers to express a heterologous cellulase and hemicellulase systemenabling cellulose and hemicellulose utilization.

Three major types of enzymatic activities are required for nativecellulose degradation: The first type are endoglucanases (1,4-β-D-glucan4-glucanohydrolases; EC 3.2.1.4). Endoglucanases cut at random in thecellulose polysaccharide chain of amorphous cellulose, generatingoligosaccharides of varying lengths and consequently new chain ends. Thesecond type are exoglucanases, including cellodextrinases(1,4-β-D-glucan glucanohydrolases; EC 3.2.1.74) and cellobiohydrolases(1,4-β-D-glucan cellobiohydrolases; EC 3.2.1.91). Exoglucanases act in aprocessive manner on the reducing or non-reducing ends of cellulosepolysaccharide chains, liberating either glucose (glucanohydrolases) orcellobiose (cellobiohydrolase) as major products. Exoglucanases can alsoact on microcrystalline cellulose, presumably peeling cellulose chainsfrom the microcrystalline structure. The third type are β-glucosidases(β-glucoside glucohydrolases; EC 3.2.1.21). β-Glucosidases hydrolyzesoluble cellodextrins and cellobiose to glucose units.

A variety of plant biomass resources are available as lignocellulosicsfor the production of biofuels, notably bioethanol. The major sourcesare (i) wood residues from paper mills, sawmills and furnituremanufacturing, (ii) municipal solid wastes, (iii) agricultural residuesand (iv) energy crops. Pre-conversion of particularly the cellulosicfraction in these biomass resources (using either physical, chemical orenzymatic processes) to fermentable sugars (glucose, cellobiose andcellodextrins) would enable their fermentation to bioethanol, providedthe necessary fermentative micro-organism with the ability to utilizethese sugars is used.

On a world-wide basis, 1.3×10¹⁰ metric tons (dry weight) of terrestrialplants are produced annually (Demain, A. L., et al., Microbiol. Mol.Biol. Rev. 69, 124-154 (2005)). Plant biomass consists of about 40-55%cellulose, 25-50% hemicellulose and 10-40% lignin, depending whether thesource is hardwood, softwood, or grasses (Sun, Y. and Cheng, J.,Bioresource Technol. 83, 1-11 (2002)). The major polysaccharide presentis water-insoluble, cellulose that contains the major fraction offermentable sugars (glucose, cellobiose or cellodextrins).

Bakers' yeast (Saccharomyces cerevisiae) remains the preferredmicro-organism for the production of ethanol (Hahn-Hägerdal, B., et al.,Adv. Biochem. Eng. Biotechnol. 73, 53-84 (2001)). Attributes that favoruse of this microbe are (i) high productivity at close to theoreticalyields (0.51 g ethanol produced/g glucose used), (ii) high osmo- andethanol tolerance, (iii) natural robustness in industrial processes,(iv) being generally regarded as safe (GRAS) due to its long associationwith wine and bread making, and beer brewing. Furthermore, S. cerevisiaeexhibits tolerance to inhibitors commonly found in hydrolyzatiesresulting from biomass pretreatment.

The major shortcoming of S. cerevisiae is its inability to utilizecomplex polysaccharides such as cellulose, or its break-down products,such as cellobiose and cellodextrins. In contrast, termites, with thehelp of microbial species that reside in their guts, are efficient atbreaking down cellulose. However, whether or not termite cellulasescould be expressed in yeast systems was not clear, as termite cellulasescould be endogenous insect cellulases or symbiotic cellulases(bacterial, protist or other). The post-translational apparatuses inyeast and insects (e.g., the glycosylation machinery) are quitedifferent, and thus it would not be expected that a termite proteincould be properly expressed in yeast. As for bacterial symbioticcellulases, it would be more predictable to express them in a bacterialhost, such as E. coli. Therefore, to address the limitations ofcurrently known bioprocessing systems, the present invention providesfor the successful heterologous expression of termite cellulases andtermite-associated symbiont cellulases in host cells, such as yeast,including Saccharomyces cerevisiae. The expression in such host cells isuseful for efficient and cost-effective consolidated bioprocessingsystems.

BRIEF SUMMARY OF THE INVENTION

The present invention provides for the heterologous expression oftermite and termite-associated symbiont cellulases in yeast cells, forexample, Saccharomyces cerevisiae.

In particular, the invention provides polynucleotides comprising anucleic acid fragment which encodes at least 50 contiguous amino acidsof a cellulase, wherein the nucleic acid fragment is codon-optimized forexpression in a yeast strain and wherein the cellulase is a termitecellulase or a termite-associated symbiont cellulase. In someembodiments, the codon adaptation index (CAI) of the nucleic acidfragment is from about 0.6 to 1.0. In some embodiments, the CAI is fromabout 0.7 to about 0.9.

In some embodiments the yeast strain can be selected from the groupconsisting of Saccharomyces cerevisiae, Kluveromyces lactus,Kluyveromyces marxianus, Schizzosaccharomyces pombe, Candida albicans,Pichia pastoris, Pichia Yarrowia lipolytica, Hansenula polymorpha,Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyceshansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe andSchwanniomyces occidentalis. In some embodiments, the yeast isSaccharomyces cerevisiae.

In further embodiments of the present invention, the cellulase hasexogluconase activity. In other embodiments, the cellulase hasendogluconase activity. In still further embodiments, the cellulase hasboth exogluconase and endogluconase activity.

In some embodiments, the cellulase is a protozoan cellulase. Thecellulase can be, for example, a Holomastigotoides mirabile,Reticulitermes speratus symbiont, Coptotermes lacteus symbiont,Reticulitermes speratus symbiont, Cryptocercus punctulatus symbiont,Mastotermes darwiniensis symbiont, Pseudotrichonympha grassii,Reticulitermes flavipes gut symbiont, Hodotermopsis sjoestedti symbiontor Neotermes koshunensis symbiont cellulase. In other embodiments, thecellulase is a metazoan cellulase. For example, the cellulase can be aCoptotermes formosanus, Nasutitermes takasagoensis, Coptotermesacinaciformis, Mastotermes darwinensis, Reticulitermes speratus,Reticulitermes flavipes, Nasutitermes walkeri or Panesthia cribratacellulase.

In other embodiments the cellulase is a bacterial cellulase, a fungalcellulase or a yeast cellulase.

In some embodiments of the invention, the polynucleotide encodes atleast about 100 contiguous amino acids of a termite cellulase or atermite-associated symbiotic cellulase. In further embodiments, thenucleic acid fragment encodes at least about 200, 300 or 350 contiguousamino acids of a cellulase.

In some embodiments, the nucleotide has additional characteristics. Forexample, in some embodiments, the polynucleotide is a polynucleotide inwhich at least one nucleotide within a sequence of 4, 5, 6, 7, 8, 9 or10 or more consecutive A, C, G or T nucleotides is replaced with adifferent nucleotide, wherein the nucleotide replacement does not alterthe amino acid sequence encoded by the polynucleotide and wherein thenucleotide replacement creates a codon that is the second mostfrequently used codon to encode an amino acid in Saccharomycescerevisiae.

In other embodiments, the polynucleotide is a polynucleotide in which atleast one restriction enzyme site within the polynucleotide is removedby replacing at least one nucleotide within the restriction enzyme sitewith a different nucleotide, wherein the nucleotide replacement does notalter the amino acid sequence encoded by the polynucleotide and whereinthe nucleotide replacement creates a codon that is the second mostfrequently used codon to encode an amino acid in Saccharomycescerevisiae. The restriction site can be, for example, a PacI, AscI,BamHI, BglII, EcoRI or XhoI restriction site.

In yet another embodiment, the polynucleotide is a polynucleotide inwhich one or more direct repeats, inverted repeats and mirror repeatswith lengths of about 5, 6, 7 8, 9 or 10 bases or longer within saidpolynucleotide is altered by replacing at least one nucleotide withinthe repeat with a different nucleotide, wherein the nucleotidereplacement does not alter the amino acid sequence encoded by thepolynucleotide and wherein the nucleotide replacement creates a codonthat is the second most frequently used codon to encode an amino acid inSaccharomyces cerevisiae.

In some embodiments, the polynucleotide of the invention is operablyassociated with a heterologous nucleic acid. For example, theheterologous nucleic acid can encode a signal peptide, and the signalpeptide can be, for example, the S. cerevisiae alpha mating factorsignal sequence. Additionally, and/or alternatively, the heterologouspolynucleotide can encode a cellulose binding domain. The cellulosebinding domain can be, for example, the cellulose binding domain of T.reesei Cbh1 or Cbh2. In some embodiments, the polynucleotide and theheterologous nucleic acid encode a fusion protein, which can be fusedfor example, via a linker sequence.

In some embodiments, the polynucleotide is at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90% orat least about 95% identical to a sequence selected from the groupconsisting of SEQ ID NO:1-20.

The invention further provides vectors comprising a polynucleotide asset forth above. The vectors can also comprise one or more additionalpolynucleotides. The one or more additional polynucleotides can encode,for example, one or more cellulases, and the one or more cellulases canbe, for example, one or more endogluconases (e.g. endogluconase I), oneor more exogluconases (e.g. cellobiohydrolase I or cellobiohydrolase II)or one or more β-glucosidases (e.g. β-glucosidase I). In someembodiments, the one or more polynucleotides can encode one or morecellulases from another organism, e.g. a T. reesei, S. fibuligera or T.emersonii cellulase. In other embodiments, the one or more additionalpolynucleotides can encode one or more additional termite ortermite-associated symbiont cellulases. In some embodiments, the one ormore additional polynucleotides can encode a cellulose binding domain.The cellulase binding domain can be, for example, the cellulose bindingdomain of T. reesei Cbh1 or Cbh2.

In some embodiments of the invention, the one or more additionalpolynucleotides in the vector can be in the forward orientation relativeto the first polynucleotide. In some embodiments, the one or moreadditional polynucleotides can be in the reverse orientation relative tothe first polynucleotide. In some embodiments, the first and additionalpolynucleotide(s) are operably associated by a linker sequence. In someembodiments, the one or more additional polynucleotides is at the 5′ endof the first polynucleotide. In some embodiments, the one or moreadditional polynucleotides is at the 3′ end of the first polynucleotide.

In some embodiments of the present invention, the vector is a plasmid.For example, the plasmid can be a yeast episomal plasmid or a yeastintegrating plasmid.

In other embodiments of the present invention the first and additionalpolynucleotides are contained in a single linear DNA construct. Thefirst and additional polynucleotides in the linear DNA construct can bein the same or different expression cassette.

The present invention also provides for host cells comprising apolynucleotide encoding at least 50 contiguous amino acids of aheterologous cellulase, wherein the heterologous cellulase is a termitecellulase or a termite-associated symbiotic cellulase, wherein the hostcell is a yeast cell and wherein the heterologous cellulase isexpressed.

The host cell can comprise a termite or termite-associated symbiontcellulase with exogluconase activity, a termite or termite-associatedsymbiont cellulase with endogluconase activity and/or a termite ortermite-associated symbiont cellulase with both exogluconase activityand endogluconase activity. In further embodiments, the host cellcomprises a termite or termite-associated symbiont cellulase withβ-glucosidase activity.

The host cells of the present invention can comprise a protozoancellulase, for example, a Holomastigotoides mirabile, Reticulitermessperatus symbiont, Coptotermes lacteus symbiont, Reticulitermes speratussymbiont, Cryptocercus punctulatus symbiont, Mastotermes darwiniensissymbiont, Pseudotrichonympha grassii, Reticulitermes flavipes gutsymbiont, Hodotermopsis sjoestedti symbiont or Neotermes koshunensissymbiont cellulase. Alternatively and/or additionally, the host cellscan also comprise a metazoan cellulase, for example a Coptotermesformosanus, Nasutitermes takasagoensis, Coptotermes acinaciformis,Mastotermes darwinensis, Reticulitermes speratus, Reticulitermesflavipes, Nasutitermes walkeri or Panesthia cribrata cellulase.

The host cells of the invention can comprise one or more cellulasesencoded by a polynucleotide comprising a sequence selected from thegroup consisting of SEQ ID NOs: 1-20. The host cells can comprise one ormore cellulases comprising the amino acid sequences of SEQ ID NOs:21-40. The host cell can contain a polynucleotide encoding a termite ortermite-associated symbiont cellulase that is codon-optimized forexpression in yeast. The host cell can also comprise a vector comprisinga polynucleotide encoding a termite or termite-associated symbiontcellulase that is codon-optimized for expression in yeast.

In some embodiments, the host cell comprises a termite ortermite-associated symbiont cellulase that is tethered to the cellsurface when expressed. In addition, the host cells can comprise atermite or termite-associated symbiont cellulase that is secreted by thecell.

In some embodiments, the host cell is a yeast selected from the groupconsisting of Saccharomyces cerevisiae, Kluveromyces lactus,Kluyveromyces marxianus, Schizzosaccharomyces pombe, Candida albicans,Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenulapolymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans,Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomycespombe and Schwanniomyces occidentalis. In one particular embodiment, theyeast is Saccharomyces cerevisiae.

In some embodiments of the present invention, the host cell comprisesone or more polynucleotides encoding one or more heterologouscellulases. The one or more polynucleotides can, for example, encode oneor more endogluconases (e.g. endogluconase I), one or more exogluconases(e.g. cellobiohydrolase I or cellobiohydrolase II) and/or one or moreβ-glucosidases (e.g. β-glucosidase I).

In some embodiments, the one or more heterologous cellulases in the hostcell is a T. reesei, S. fibuligera and/or T. emersonii cellulase. Inaddition, the one or more heterologous cellulases can be a termitecellulase or a termite-associated symbiont cellulase. In someembodiments, the one or more heterologous cellulases is encoded by apolynucleotide selected from the polynucleotides of SEQ ID NOs: 1-20. Insome embodiments, the one or more heterologous cellulases is a proteinwhich comprises an amino acid sequence selected from SEQ ID NOs: 21-40.

In other aspects the invention encompasses host cells comprising one ormore termite cellulases or termite-associate symbiont cellulases whereinat least one heterologous cellulase is tethered to the cell surface whenexpressed. In other embodiments, at least one heterologous cellulase issecreted by the cell. In still further embodiments, at least oneheterologous cellulase is tethered to the cell surface and at least oneheterologous cellulase is secreted by the cell.

The invention also provides for host cells, wherein the host cells havethe ability to saccharify crystalline cellulose. In additionalembodiments, the host cells also have the ability to ferment crystallinecellulose.

Furthermore, the invention provides methods of using thepolynucleotides, vectors, polypeptides and host cells of the invention.For example, the invention provides a method for hydrolyzing acellulosic substrate, comprising contacting the cellulosic substratewith a host cell of the invention. In some embodiments, the cellulosicsubstrate comprises a lignocellulosic biomass selected from the groupconsisting of grass, switch grass, cord grass, rye grass, reed canarygrass, miscanthus, sugar-processing residues, sugarcane bagasse,agricultural wastes, rice straw, rice hulls, barley straw, corn cobs,cereal straw, wheat straw, canola straw, oat straw, oat hulls, cornfiber, stover, soybean stover, corn stover, forestry wastes, recycledwood pulp fiber, paper sludge, sawdust, hardwood, softwood, andcombinations thereof.

In addition, the invention also provides a method of fermentingcellulose using the host cells of the invention. The method comprisesculturing a host cell in medium that contains crystalline celluloseunder suitable conditions for a period sufficient to allowsaccharification and fermentation of the cellulose. In still furtherembodiments, the host cell produces ethanol.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 depicts a plasmid map of pMU451. Synthetic termite cellulasegenes were inserted into the PacI/AscI sites. “S.cer ENO1 pr” and “S.cerENO ter” indicate the S. cerevisiae ENO1 promoter and terminatorsequences respectively. “S.cer URA3” indicates the S. cerevisiae URA3auxotrophic marker. “2 mu ori” indicates the S. cerevisiae 2 mu plasmidorigin of replication sequence. “Bla(AmpR)” indicates the Amp resistancesequence, and “pBR322 ori” indicates the E. coli pB322 plasmid origin ofreplication sequence.

FIG. 2 depicts a bar graph showing Avicel conversion by supernatants ofS. cerevisiae strains expressing termite cellulase genes. “Straincontrol” indicates MO375 strain transformed with empty pMU451 vector(negative control). “T.r.EG1” indicates MO375 transformed with T. reeseiendogluconase 1 (EG1) in pMU451 vector (positive control). Numbering ofother strains is according to numbering shown in Table 5. “Buffercontrol” indicates the condition in which Avicel conversion assayreaction buffer was used instead of yeast culture supernatant.

FIG. 3 depicts an image of a Congo Red test performed on S. cerevisiaeexpressing termite cellulase or termite-associated symbiotic protozoancellulase genes. “MO419” indicates MO375 strain transformed with emptypMU451 vector (negative control). “MO423” indicates MO375 transformedwith T. reesei EG1 in pMU451 vector (positive control). “MO247”indicates Y294 strain with furl gene knocked out (to stabilize theepisomal plasmid) and expressing T. emersonii CBH1 in a vector similarto pMU451. “MO449” corresponds to MO375 transformed with Coptotermesformosanus EG (CfEG). Numbering of other strains is according tonumbering shown in Table 5.

FIG. 4 depicts a bar graph showing results of a carboxymethyl-cellulose(CMC) conversion assay using S. cerevisiae expressing termite cellulasegenes. “MO419” indicates MO375 strain transformed with empty vector(negative control). “MO423” indicates MO375 transformed with T. reeseiEG1 (positive control). Numbering of other strains is according tonumbering shown in Table 5. “Buffer control” indicates the condition inwhich buffer was used instead of yeast culture supernatant.

FIG. 5 depicts a bar graph showing the effect of addition of yeast madetermite CfEG to yeast made fungal CBHs on crystalline celluloseconversion measured by Avicel assay. “Neg Cont” is the negative controland corresponds to 300 μl of parental non-cellulytic M0509 strainsupernatant; “CBH” corresponds to 100 ul of CBH mix (M0579 and M0969samples mixed at ratio 4:1) added to 200 μl of control M0509supernatant; “CBH+EG” corresponds to 100 μl of CBH mix added to 200 μlof M0968 supernatant (CfEG). All measurements were done inquadruplicates. The samples and strains are also described in Table 6below.

FIG. 6 depicts a graph showing the results of SSF ethanol production ofco-cultured cellulytic yeast strains at different external enzyme loadscompared to the control non-cellulytic strain MO249. 100% of externalcellulase load corresponds to 10 mg of enzyme per gram of Avicel.

FIG. 7 depicts a graph showing the theoretical ethanol yield at 160 hrsof SSF plotted against external cellulase loads. The co-culture containsstrains MO595, 563, 592, 566; MO249 is the control non-cellulyticstrain.

FIG. 8 depicts a graph showing results for cellulase production by yeastin a bioreactor. Strain M0712 was batch cultivated in YPD-based richmedia with 50 g/L, glucose in 1 L bioreactor for 24 hours, followed by astepped feed of 50% glucose with vitamins and trace elements for another36 hours. At several time points, reactor samples were taken and the drycell weight was measured. Additionally, the protein concentration foreach cellulase was measured by HPLC.

FIG. 9 depicts a graph showing results from an Avicel conversion assayutilizing supernatants of S. cerevisiae strains expressing termitecellulase genes in synergy with a yeast-made T. emersonii CBH1 (strainM0420). “NegCont” corresponds to the negative control MO375 straintransformed with empty pMU451 vector. “MO423” corresponds to the MO375strain transformed with T. reesei endogluconase 1 (EG1) in the pMU451vector (positive control). The other numbered strains are described inTable 5 below. For single strains, 300 μl or supernatant was used; forthe combined samples 150 μl of each supernatant was used. For example,“420/423” means that 150 μl of the M0420 strain supernatant was mixedwith 150 μl of the M0423 supernatant.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to, inter alia, the heterologousexpression of termite cellulases and termite-associated symbiontcellulases in host cells, including yeast, e.g., Saccharomycescerevisiae. The present invention provides important tools to enablegrowth of yeast on cellulosic substrates for ethanol production.

DEFINITIONS

A “vector,” e.g., a “plasmid” or “YAC” (yeast artificial chromosome)refers to an extrachromosomal element often carrying one or more genesthat are not part of the central metabolism of the cell, and is usuallyin the form of a circular double-stranded DNA molecule. Such elementsmay be autonomously replicating sequences, genome integrating sequences,phage or nucleotide sequences, linear, circular, or supercoiled, of asingle- or double-stranded DNA or RNA, derived from any source, in whicha number of nucleotide sequences have been joined or recombined into aunique construction which is capable of introducing a promoter fragmentand DNA sequence for a selected gene product along with appropriate 3′untranslated sequence into a cell. Preferably, the plasmids or vectorsof the present invention are stable and self-replicating.

An “expression vector” is a vector that is capable of directing theexpression of genes to which it is operably associated.

The term “heterologous” as used herein refers to an element of a vector,plasmid or host cell that is derived from a source other than theendogenous source. Thus, for example, a heterologous sequence could be asequence that is derived from a different gene or plasmid from the samehost, from a different strain of host cell, or from an organism of adifferent taxonomic group (e.g., different kingdom, phylum, class,order, family genus, or species, or any subgroup within one of theseclassifications). The term “heterologous” is also used synonymouslyherein with the term “exogenous.”

The term “domain” as used herein refers to a part of a molecule orstructure that shares common physical or chemical features, for examplehydrophobic, polar, globular, helical domains or properties, e.g., a DNAbinding domain or an ATP binding domain. Domains can be identified bytheir homology to conserved structural or functional motifs. Examples ofcellulase domains include the catalytic domain (CD) and the cellulosebinding domain (CBD).

A “nucleic acid,” “polynucleotide,” or “nucleic acid molecule” is apolymeric compound comprised of covalently linked subunits callednucleotides. Nucleic acid includes polyribonucleic acid (RNA) andpolydeoxyribonucleic acid (DNA), both of which may be single-stranded ordouble-stranded. DNA includes cDNA, genomic DNA, synthetic DNA, andsemi-synthetic DNA.

An “isolated nucleic acid molecule” or “isolated nucleic acid fragment”refers to the phosphate ester polymeric form of ribonucleosides(adenosine, guanosine, uridine or cytidine; “RNA molecules”) ordeoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, ordeoxycytidine; “DNA molecules”), or any phosphoester analogs thereof,such as phosphorothioates and thioesters, in either single strandedform, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA andRNA-RNA helices are possible. The term nucleic acid molecule, and inparticular DNA or RNA molecule, refers only to the primary and secondarystructure of the molecule, and does not limit it to any particulartertiary forms. Thus, this term includes double-stranded DNA found,inter alia, in linear or circular DNA molecules (e.g., restrictionfragments), plasmids, and chromosomes. In discussing the structure ofparticular double-stranded DNA molecules, sequences may be describedherein according to the normal convention of giving only the sequence inthe 5′ to 3′ direction along the non-transcribed strand of DNA (i.e.,the strand having a sequence homologous to the mRNA).

A “gene” refers to an assembly of nucleotides that encode a polypeptide,and includes cDNA and genomic DNA nucleic acids. “Gene” also refers to anucleic acid fragment that expresses a specific protein, includingintervening sequences (introns) between individual coding segments(exons), as well as regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength. Hybridization and washing conditions are well known andexemplified, e.g., in Sambrook, J., Fritsch, E. F. and Maniatis, T.MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter11 and Table 11.1 therein (hereinafter “Maniatis”, entirely incorporatedherein by reference). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization. Stringency conditionscan be adjusted to screen for moderately similar fragments, such ashomologous sequences from distantly related organisms, to highly similarfragments, such as genes that duplicate functional enzymes from closelyrelated organisms. Post-hybridization washes determine stringencyconditions. One set of conditions uses a series of washes starting with6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with0.2×SSC, 0.5% SDS at 50° C. for 30 min. For more stringent conditions,washes are performed at higher temperatures in which the washes areidentical to those above except for the temperature of the final two 30min washes in 0.2×SSC, 0.5% SDS are increased to 60° C. Another set ofhighly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDSat 65° C. An additional set of highly stringent conditions are definedby hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC,0.1% SDS followed by 0.1×SSC, 0.1% SDS.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (see, e.g.,Maniatis at 9.50-9.51). For hybridizations with shorter nucleic acids,i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see, e.g., Maniatis, at 11.7-11.8). In one embodiment thelength for a hybridizable nucleic acid is at least about 10 nucleotides.Preferably a minimum length for a hybridizable nucleic acid is at leastabout 15 nucleotides; more preferably at least about 20 nucleotides; andmost preferably the length is at least 30 nucleotides. Furthermore, theskilled artisan will recognize that the temperature and wash solutionsalt concentration may be adjusted as necessary according to factorssuch as length of the probe.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences.

As known in the art, “similarity” between two polypeptides is determinedby comparing the amino acid sequence and conserved amino acidsubstitutes thereto of the polypeptide to the sequence of a secondpolypeptide.

Suitable nucleic acid sequences or fragments thereof (isolatedpolynucleotides of the present invention) encode polypeptides that areat least about 70% to 75% identical to the amino acid sequences reportedherein, at least about 80%, 85%, or 90% identical to the amino acidsequences reported herein, or at least about 95%, 96%, 97%, 98%, 99%, or100% identical to the amino acid sequences reported herein. Suitablenucleic acid fragments are at least about 70%, 75%, or 80% identical tothe nucleic acid sequences reported herein, at least about 80%, 85%, or90% identical to the nucleic acid sequences reported herein, or at leastabout 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acidsequences reported herein. Suitable nucleic acid fragments not only havethe above identities/similarities but typically encode a polypeptidehaving at least 50 amino acids, at least 100 amino acids, at least 150amino acids, at least 200 amino acids, or at least 250 amino acids.

The term “probe” refers to a single-stranded nucleic acid molecule thatcan base pair with a complementary single stranded target nucleic acidto form a double-stranded molecule.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable to hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine. Accordingly, the instant inventionalso includes isolated nucleic acid fragments that are complementary tothe complete sequences as reported in the accompanying Sequence Listingas well as those substantially similar nucleic acid sequences.

As used herein, the term “oligonucleotide” refers to a nucleic acid,generally of about 18 nucleotides, that is hybridizable to a genomic DNAmolecule, a cDNA molecule, or an mRNA molecule. Oligonucleotides can belabeled, e.g., with 32P-nucleotides or nucleotides to which a label,such as biotin, has been covalently conjugated. An oligonucleotide canbe used as a probe to detect the presence of a nucleic acid according tothe invention. Similarly, oligonucleotides (one or both of which may belabeled) can be used as PCR primers, either for cloning full length or afragment of a nucleic acid of the invention, or to detect the presenceof nucleic acids according to the invention. Generally, oligonucleotidesare prepared synthetically, preferably on a nucleic acid synthesizer.Accordingly, oligonucleotides can be prepared with non-naturallyoccurring phosphoester analog bonds, such as thioester bonds, etc.

A DNA or RNA “coding region” is a DNA or RNA molecule which istranscribed and/or translated into a polypeptide in a cell in vitro orin vivo when placed under the control of appropriate regulatorysequences. “Suitable regulatory regions” refer to nucleic acid regionslocated upstream (5′ non-coding sequences), within, or downstream (3′non-coding sequences) of a coding region, and which influence thetranscription, RNA processing or stability, or translation of theassociated coding region. Regulatory regions may include promoters,translation leader sequences, RNA processing site, effector binding siteand stem-loop structure. The boundaries of the coding region aredetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxyl) terminus. A coding region can include,but is not limited to, prokaryotic regions, cDNA from mRNA, genomic DNAmolecules, synthetic DNA molecules, or RNA molecules. If the codingregion is intended for expression in a eukaryotic cell, apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding region.

“Open reading frame” is abbreviated ORF and means a length of nucleicacid, either DNA, cDNA or RNA, that comprises a translation start signalor initiation codon, such as an ATG or AUG, and a termination codon andcan be potentially translated into a polypeptide sequence.

“Promoter” refers to a DNA fragment capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingregion is located 3′ to a promoter. Promoters may be derived in theirentirety from a native gene, or be composed of different elementsderived from different promoters found in nature, or even comprisesynthetic DNA segments. It is understood by those skilled in the artthat different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity. A promoter isgenerally bounded at its 3′ terminus by the transcription initiationsite and extends upstream (5′ direction) to include the minimum numberof bases or elements necessary to initiate transcription at levelsdetectable above background. Within the promoter will be found atranscription initiation site (conveniently defined for example, bymapping with nuclease Si), as well as protein binding domains (consensussequences) responsible for the binding of RNA polymerase.

A coding region is “under the control” of transcriptional andtranslational control elements in a cell when RNA polymerase transcribesthe coding region into mRNA, which is then trans-RNA spliced (if thecoding region contains introns) and translated into the protein encodedby the coding region.

“Transcriptional and translational control regions” are DNA regulatoryregions, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding region in a host cell. Ineukaryotic cells, polyadenylation signals are control regions.

The term “operably associated” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably associatedwith a coding region when it is capable of affecting the expression ofthat coding region (i.e., that the coding region is under thetranscriptional control of the promoter). Coding regions can be operablyassociated to regulatory regions in sense or antisense orientation.

The term “expression,” as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

Termite Cellulases and Termite-Associated Symbiont Cellulases

Termite guts have been referred to as tiny bioreactors due to theirefficiency at lignocellulose digestion. This efficiency can beattributed not only to the activity of cellulases encoded by the termitegenome, but also to the microbes that populate termite guts. The presentinvention provides for the use of both termite cellulases (i.e.cellulases that are expressed endogenously in termite cells) andtermite-associated symbiont cellulases (i.e. cellulases that areexpressed by symbiotic organisms found in termite guts).

In some embodiments of the present invention, the cellulase is a termitecellulase. The termite can be, for example, a higher termite, i.e. atermite from the family Termitidae. The termite of can also be a lowertermite. For example, the lower termite can be a Mastotermiitidae,Hodotermitidae, Termopsidae, Kalotermitidae, Rhinotermitidae orSerritermitidae. In some embodiments, the termite is selected from thegroup consisting of Coptotermes formosanus, Nasutitermes takasagoensis,Coptotermes acinaciformis, Mastotermes darwinensis, Reticulitermessperatus, Reticulitermes flavipes, Nasutitermes walkeri and Panesthiacribrata.

According to the present invention, the cellulase can also be from atermite-associated symbiont. The termite-associated symbiont can be, forexample, a fungal symbiont, a yeast symbiont, a bacterial symbiont or aprotozoan symbiont. The bacterial symbiont can be, for example,fibroacters or spirochetes. The protozoan symbiont can be, for example,a flagellated protozoan. In some embodiments, the protozoan symbiont isan actinomycete. In some embodiments, the protozoan symbiont is selectedfrom the group consisting of Holomastigotoides mirabile, Reticulitermessperatus symbiont, Coptotermes lacteus symbiont, Reticulitermes speratussymbiont, Cryptocercus punctulatus symbiont, Mastotermes darwiniensissymbiont, Pseudotrichonympha grassii, Reticulitermes flavipes gutsymbiont, Hodotermopsis sjoestedti symbiont and Neotermes koshunensissymbiont.

In some embodiments of the present invention, the cellulase hasendogluconase activity. In some embodiments, the cellulase hasexogluconase activity. In some embodiments, the cellulase has bothexogluconase and endogluconase activity. In some embodiments of theinvention, the cellulase has β-glucosidase activity. Endogluconase,exogluconase and β-glucosidase activity can be determined using anymethod known in the art. For example, CMC conversion assays are commonlyused to assess endogluconase activity, and Avicel conversion assays arecommonly used to assess exogluconase activity.

Codon Optimization

According to the present invention, sequences encoding cellulases can becodon optimized. As used herein the term “codon optimized” refers to anucleic acid that has been adapted for expression in the cells of agiven organism by replacing at least one, or more than one, or asignificant number, of codons with one or more codons that are morefrequently used in the genes of that organism.

In general, highly expressed genes in an organism are biased towardscodons that are recognized by the most abundant tRNA species in thatorganism. One measure of this bias is the “codon adaptation index” or“CM,” which measures the extent to which the codons used to encode eachamino acid in a particular gene are those which occur most frequently ina reference set of highly expressed genes from an organism. The CodonAdaptation Index is described in more detail in Sharp and Li, NucleicAcids Research 15: 1281-1295 (1987)), which is incorporated by referenceherein in its entirety.

The CAI of codon optimized sequences of the present invention can befrom about 0.5 to 1.0, from about 0.6 to 1.0, from about 0.7 to 1.0,from about 0.75 to 1.0, from about 0.8 to 1.0 or from about 0.9 to 1.0.In some embodiments, the CM of the codon optimized sequences of thepresent invention corresponds to from about 0.5 to about 0.9, from about0.7 to about 0.9, from about 0.6 to about 0.8, from about 0.7 to about0.8 or from about 0.75 to about 0.8.

A codon optimized sequence may be further modified for expression in aparticular organism, depending on that organism's biologicalconstraints. For example, large runs of “As” or “Ts” (e.g., runs greaterthan 3, 4, 5, 6, 7, 8, 9, or 10 consecutive bases) can effecttranscription negatively. Therefore, it can be useful to remove a runby, for example, replacing at least one nucleotide in the run withanother nucleotide. Furthermore, specific restriction enzyme sites maybe removed for molecular cloning purposes by replacing at least onenucleotide in the restriction site with another nucleotide. Examples ofsuch restriction enzyme sites include PacI, AscI, BamHI, BglII, EcoRIand XhoI. Additionally, the DNA sequence can be checked for directrepeats, inverted repeats and mirror repeats with lengths of about 5, 6,7, 8, 9 or 10 bases or longer. Runs of “As” or “Ts”, restriction sitesand/or repeats can be modified by replacing at least one codon withinthe sequence with the “second best” codons, i.e., the codon that occursat the second highest frequency for a particular amino acid within theparticular organism for which the sequence is being optimized.

Deviations in the nucleotide sequence that comprise the codons encodingthe amino acids of any polypeptide chain allow for variations in thesequence coding for the gene. Since each codon consists of threenucleotides, and the nucleotides comprising DNA are restricted to fourspecific bases, there are 64 possible combinations of nucleotides, 61 ofwhich encode amino acids (the remaining three codons encode signalsending translation). The “genetic code” which shows which codons encodewhich amino acids is reproduced herein as Table 1. As a result, manyamino acids are designated by more than one codon. For example, theamino acids alanine and proline are coded for by four triplets, serineand arginine by six triplets each, whereas tryptophan and methionine arecoded for by just one triplet. This degeneracy allows for DNA basecomposition to vary over a wide range without altering the amino acidsequence of the proteins encoded by the DNA.

TABLE 1 The Standard Genetic Code T C A G T TTT Phe (F) TCT Ser (S)TAT Tyr (Y) TGT Cys (C) TTC Phe (F) TCC Ser (S) TAC Tyr (Y) TGCTTA Leu (L) TCA Ser (S) TAA Ter TGA Ter TTG Leu (L) TCG Ser (S) TAG TerTGG Trp (W) C CTT Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R)CTC Leu (L) CCC Pro (P) CAC His (H) CGC Arg (R) CTA Leu (L) CCA Pro (P)CAA Gln (Q) CGA Arg (R) CTG Leu (L) CCG Pro (P) CAG Gln (Q) CGG Arg (R)A ATT Ile (I) ACT Thr (T) AAT Asn (N) AGT Ser (S) ATC Ile (I)ACC Thr (T) AAC Asn (N) AGC Ser (S) ATA Ile (I) ACA Thr (T) AAA Lys (K)AGA Arg (R) ATG Met (M) ACG Thr (T) AAG Lys (K) AGG Arg (R) GGTT Val (V) GCT Ala (A) GAT Asp (D) GGT Gly (G) GTC Val (V) GCC Ala (A)GAC Asp (D) GGC Gly (G) GTA Val (V) GCA Ala (A) GAA Glu (E) GGA Gly (G)GTG Val (V) GCG Ala (A) GAG Glu (E) GGG Gly (G)

Many organisms display a bias for use of particular codons to code forinsertion of a particular amino acid in a growing peptide chain. Codonpreference or codon bias, differences in codon usage between organisms,is afforded by degeneracy of the genetic code, and is well documentedamong many organisms. Codon bias often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, inter alia, the properties of the codons being translatedand the availability of particular transfer RNA (tRNA) molecules. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based oncodon optimization.

Given the large number of gene sequences available for a wide variety ofanimal, plant and microbial species, it is possible to calculate therelative frequencies of codon usage. Codon usage tables are readilyavailable, for example, athttp://phenotype.biosci.umbc.edu/codon/sgd/index.php (visited May 7,2008) or at http://www.kazusa.or.jp/codon/ (visited Mar. 20, 2008), andthese tables can be adapted in a number of ways. See Nakamura, Y., etal. “Codon usage tabulated from the international DNA sequencedatabases: status for the year 2000” Nucl. Acids Res. 28:292 (2000).Codon usage tables for yeast, calculated from GenBank Release 128.0 [15Feb. 2002], are reproduced below as Table 2. This table uses mRNAnomenclature, and so instead of thymine (T) which is found in DNA, thetables use uracil (U) which is found in RNA. The Table has been adaptedso that frequencies are calculated for each amino acid, rather than forall 64 codons.

TABLE 2 Codon Usage Table for Saccharomyces  cerevisiae Genes FrequencyAmino per Acid Codon Number hundred Phe UUU 170666 26.1 Phe UUC 12051018.4 Leu UUA 170884 26.2 Leu UUG 177573 27.2 Leu CUU  80076 12.3 Leu CUC 35545 5.4 Leu CUA  87619 13.4 Leu CUG  68494 10.5 Ile AUU 196893 30.1Ile AUC 112176 17.2 Ile AUA 116254 17.8 Met AUG 136805 20.9 Val GUU144243 22.1 Val GUC  76947 11.8 Val GUA  76927 11.8 Val GUG  70337 10.8Ser UCU 153557 23.5 Ser UCC  92923 14.2 Ser UCA 122028 18.7 Ser UCG 55951 8.6 Ser AGU  92466 14.2 Ser AGC  63726 9.8 Pro CCU  88263 13.5Pro CCC  44309 6.8 Pro CCA 119641 18.3 Pro CCG  34597 5.3 Thr ACU 13252220.3 Thr ACC  83207 12.7 Thr ACA 116084 17.8 Thr ACG  52045 8.0 Ala GCU138358 21.2 Ala GCC  82357 12.6 Ala GCA 105910 16.2 Ala GCG  40358 6.2Tyr UAU 122728 18.8 Tyr UAC  96596 14.8 His CAU  89007 13.6 His CAC 50785 7.8 Gln CAA 178251 27.3 Gln CAG  79121 12.1 Asn AAU 233124 35.7Asn AAC 162199 24.8 Lys AAA 273618 41.9 Lys AAG 201361 30.8 Asp GAU245641 37.6 Asp GAC 132048 20.2 Glu GAA 297944 45.6 Glu GAG 125717 19.2Cys UGU  52903 8.1 Cys UGC  31095 4.8 Trp UGG  67789 10.4 Arg CGU  417916.4 Arg CGC  16993 2.6 Arg CGA  19562 3.0 Arg CGG  11351 1.7 Arg AGA139081 21.3 Arg AGG  60289 9.2 Gly GGU 156109 23.9 Gly GGC  63903 9.8Gly GGA  71216 10.9 Gly GGG  39359 6.0 Stop UAA   6913 1.1 Stop UAG  3312 0.5 Stop UGA   4447 0.7

By utilizing this or similar tables, one of ordinary skill in the artcan apply the frequencies to any given polypeptide sequence, and producea nucleic acid fragment of a codon-optimized coding region which encodesthe polypeptide, but which uses codons optimal for a given species.Codon-optimized coding regions can be designed by various differentmethods.

In one method, a codon usage table is used to find the single mostfrequent codon used for any given amino acid, and that codon is usedeach time that particular amino acid appears in the polypeptidesequence. For example, referring to Table 2 above, for leucine, the mostfrequent codon is UUG, which is used 27.2% of the time. Thus, suing thismethod, all the leucine residues in a given amino acid sequence would beassigned the codon UUG.

In another method, a codon-optimized sequence contains the samefrequency of each codon as is used in the organism where thecodon-optimized sequence is intended to be expressed. Thus, using thismethod for optimization, if a hypothetical polypeptide sequence had 100leucine residues, referring to Table 2 for frequency of usage in the S.cerevisiae, about 5, or 5% of the leucine codons would be CUC, about 11,or 11% of the leucine codons would be CUG, about 12, or 12% of theleucine codons would be CUU, about 13, or 13% of the leucine codonswould be CUA, about 26, or 26% of the leucine codons would be UUA, andabout 27, or 27% of the leucine codons would be UUG. Using this method,the frequency of codon usage, and not necessarily the order of thecodons, is important. Thus, as will be understood by those of ordinaryskill in the art, the distribution of codons in the sequence can varysignificantly using this method. However, the sequence always encodesthe same polypeptide.

In one embodiment of the invention, a sequence can be codon-optimizedfor expression in two yeast strains, for example, in both Saccharomycescerevisiae and Kluveromyces lactus. Thus, according to this embodiment,codons are selected according to their usage in both strains.

Codon-optimized sequences of the present invention include those as setforth in Table 3 below:

TABLE 3 Termite cellulase genes constructed Donor Accession number andorganism DNA sequence used amino acid sequence Holomasti-TTAATTAAAATGAGATTTCCATCTATTTTCACTGCTGTTTTGTTCGCAGCCTCAAGTGCAccession No: AB071011 gotoidesTTTAGCAGAGAAACATCCTAAGTTCGTATGGCAACAGTGTACAAAGGGTGGATGCTCMLVALAVSVFCEKHPKFVWQQCTK mirabileCGACGTGTCTGGTTATTTGGTGCACGACAGACATATAGGGGACGTTTGGGACAGAGAGGCSDVSGYLVHDRHIGDVWDRENGAATACCGATTACCCAGAATTAGATTACGACGCCAACGTAGGTGTCACTGTGTCAGCTDYPELDYDANVGVTVSADGKTLSAGACGGAAAGACTTTATCACAAAGATTAGTTTCCAAATTGTGGGACGATAAGAAAGCQRLVSKLWDDKKAVGSRVYIVDTTAGTAGGATCTAGAGTGTACATAGTGGACACCACTGACAAGAAATATCAGTTATTTCADKKYQLFQFVGKEFTYTVDMSQIPCATTTGTTGGTAAGGAGTTTACATACACTGTGGACATGTCACAAATTCCTTGCGGTGTCGVNAALYTVEMPAEGKSPGGVEYGAACGCCGCTTTGTACACTGTCGAAATGCCAGCAGAGGGAAAATCTCCTGGTGGTGTAYGYCDANCVDGGCCMEFDIQEASSGAATACGGTTATGGTTACTGCGACGCAAACTGTGTGGACGGTGGATGTTGCATGGAGKAIVYTTHSCQSQTGGCDTSGCGYNTTCGATATCCAAGAAGCCTCCTCTAAGGCAATAGTGTACACAACTCACTCATGTCAATPYRDSNDHAFWGQTINVNQPVTIVTCTCAGACCGGAGGTTGCGACACAAGTGGTTGTGGTTACAACCCATATAGAGATTCAAQFVGSGGSLTEVKRLYVQGGKVTPATGACCACGCCTTTTGGGGTCAAACTATTAATGTCAACCAACCTGTGACTATAGTGACAAKSLSDSYCNVNDYRSLKTIGASFACAGTTCGTTGGATCAGGTGGTTCTTTAACTGAAGTCAAGAGATTGTACGTCCAAGGQRGHVVVFSLWDSDGMSWMDGGNAGGTAAAGTGACCCCAGCAGCCAAAAGTTTATCCGATTCATATTGCAATGTTAACGAAGPCTSYNVATVESSQPNLKVTWSNCTATCGTTCTTTGAAAACAATAGGAGCTTCATTCCAAAGAGGACATGTAGTCGTGTTC VKFGDIDSTYTCATTATGGGACAGTGATGGAATGTCCTGGATGGATGGTGGAAACGCCGGTCCTTGT(SEQ ID NO: 21)ACGAGTTACAACGTTGCAACCGTTGAATCATCTCAGCCAAATTTGAAAGTAACATGGTCCAACGTCAAGTTTGGTGATATCGACAGTACTTACTAAGGCGCGCC (SEQ ID NO: 1)Coptotermes TTAATTAAAATGAGATTTCCTTCCATATTCACCGCTGTTTTGTTCGCAGCCTCAAGTGCAccession No: AB089801 lacteusTTTAGCAGAATGTACTAAGGGTGGATGTACTAACAAGAATGGATACATAGTTCATGAECTKGGCTNKNGYIVHDKHVGDIQ symbiontTAAGCACGTCGGTGACATCCAGAATAGAGACACTTTGGACCCTCCAGACTTAGATTANRDTLDPPDLDYEKDVGVTVSGGTLTGAAAAGGACGTGGGAGTAACCGTGTCCGGTGGAACCCTTAGTCAAAGATTAGTCTCSQRLVSTWNGKKVVGSRLYIVDEAAACTTGGAACGGTAAGAAAGTCGTGGGAAGTAGATTGTATATTGTGGACGAAGCCGADEKYQLFTFVGKEFTYTVDMSQIQCCGAGAAATATCAATTATTCACATTTGTCGGTAAGGAGTTCACCTATACCGTTGATATGGINAALYTVEMPAAGKTPGGVKYGTCCCAGATCCAATGTGGAATCAATGCCGCATTATACACAGTGGAAATGCCTGCCGCTYGYCDANCVDGDCCMEFDIQEASNGGAAAGACCCCTGGAGGTGTTAAGTATGGATATGGATATTGTGATGCCAACTGCGTGKAIVYTTHSCQSQTSGCDTSGCGYNGATGGAGATTGTTGTATGGAGTTCGATATCCAAGAAGCTTCTAACAAGGCAATCGTTPYRDSGDKAFWGTTINVNQPVTIVTTACACCACCCATTCCTGTCAAAGTCAAACTTCAGGTTGCGATACCTCAGGATGCGGTTQFIGSGSSLTEVKRLCVQGGKTFPPAACAACCCTTACAGAGACAGTGGTGACAAGGCATTCTGGGGAACAACTATAAACGTAAKSLTDSYCNANDYRSLRTMGASMAACCAGCCTGTGACAATTGTAACACAGTTTATCGGTTCTGGTAGTTCCTTAACTGAAGTRGHVVVFSLWDSNGMSWMDGGNACAAAAGATTGTGCGTGCAAGGTGGAAAGACCTTCCCTCCAGCCAAATCATTAACCGAGPCTSYNIESLESSQPNLKVTWSNVKCAGTTATTGTAATGCCAACGACTATAGAAGTTTGAGAACTATGGGTGCATCCATGGC YGEIDSPYTAGAGGACACGTTGTTGTGTTTTCTTTGTGGGATTCTAATGGTATGAGTTGGATGGAT(SEQ ID NO: 22)GGAGGTAACGCCGGTCCTTGTACCTCATATAATATTGAATCTTTGGAATCCAGTCAGCCAAACTTAAAGGTCACATGGTCAAACGTGAAATACGGAGAGATCGATTCTCCTTATT AAGGCGCGCC(SEQ ID NO: 2) CryptocercusTTAATTAAAATGAGATTTCCATCTATTTTCACTGCTGTGTTGTTTGCCGCTTCAAGTGCAccession No: AB274702 punctulatusTTTAGCATCTAGAATATCCGTGTCATGGTTGAGTACATCCGGTTCCAAAATAACCGATMLLFLLSRISVSWLSTSGSKITDGGQ symbiontGGAGGTCAAACTGTCAGATTAACAGGAGTGAATTGGTTTGGTTATGAAACCTCAGAGTVRLTGVNWFGYETSEEVFHGLWAGAAGTGTTTCACGGTTTGTGGGCCGCTGGTTTGCACGACTTGGTACAGGGTGTCTCCCAGLHDLVQGVSQKKFNTFRVPISASAAAAGAAATTCAACACTTTTAGAGTGCCTATTTCCGCATCTGTTTTGCAAGACTGGAAVLQDWKAGKPNPKPNINLNVNADLGGCCGGAAAGCCAAACCCAAAACCAAACATCAATTTGAACGTGAATGCTGACTTAGAEGLNNQQIFDLFLADCKKYKIYVFIDGGGTTTGAACAATCAACAAATATTCGACTTATTCTTAGCCGACTGTAAGAAGTACAAVHGVTDGSYMDNLWYTSAHPAEWIAATCTACGTGTTCATCGACGTGCATGGTGTTACAGATGGATCATATATGGACAACTTAYSALEWFADHYKGDQTIIGIDIKNEPTGGTACACCTCTGCTCACCCTGCCGAATGGATATACAGTGCATTGGAGTGGTTCGCCGHGRCEQAEAAKWSDSKDNNNWKYATCACTACAAGGGAGATCAGACTATTATAGGTATTGACATAAAGAACGAGCCACACGFIETAAARILGKNPNLLILVEGIECYNGTAGATGCGAACAAGCCGAAGCAGCTAAGTGGTCCGATAGTAAAGACAATAATAACNNWGWWGGNLIPVNDYPINLGSGQTGGAAGTACTTCATTGAGACAGCCGCAGCTAGAATCTTAGGTAAGAATCCTAACTTGKQLVYSPHEYGPSVNDQSWFKSGFNTTAATATTGGTTGAAGGAATTGAGTGTTACAACAACAACTGGGGTTGGTGGGGTGGAYDSLYADHWQKMWMFIIEKNIAPILAACTTAATCCCAGTTAATGACTATCCTATAAACTTGGGTTCTGGACAGAAGCAATTAGIGEWGGHVVEPNTTWMKALVQLISTCTATTCCCCACACGAATACGGTCCTTCTGTGAATGATCAGTCATGGTTCAAATCTGGKYGLSQTFWCLNPDSGDTGGLLENTTTCAATTATGATTCCTTGTACGCCGATCATTGGCAAAAGATGTGGATGTTCATTATCDWITWDTAKLDIIKGVLGAAAAGAACATCGCCCCTATATTGATCGGAGAGTGGGGTGGTCACGTTGTAGAACCT(SEQ ID NO: 23)AATACTACCTGGATGAAGGCTTTGGTCCAATTAATATCCAAATATGGATTGTCACAAACTTTCTGGTGCTTAAACCCTGATAGTGGTGACACTGGAGGTTTGTTAGAAAACGATTGGATAACTTGGGATACAGCCAAATTGGATATAATTAAAGGTGTGTTATAAGGCGCGC C(SEQ ID NO: 3) MastotermesTTAATTAAAATGAGATTTCCATCTATTTTCACTGCTGTTTTGTTCGCAGCCTCAAGTGCAccession No: AB274656 darwiniensisTTTAGCAGCCTATTACATCTCCGCTTCTGGTAATGAGTTGGTGGACCCAACCGGAAAAMLVLLASFGVAYYISASGNELVDPT symbiontCAATTAAGAATCACCGGTATAAACTGGTTTGGATTCGAGACTTCACAGTCTGCTTTTCGKQLRITGINWFGFETSQSAFHGLWACGGTTTGTGGAACGCCAACTTACACAAGGTCGTGCAACAGGTTGCGGAGCACGGTTNANLHKVVQQVAEHGFNCFRCPISCTTAATTGCTTCAGATGTCCAATCTCCTGTGACTTGATCCACAAATGGATGAGAGGAGADLIHKWMRGDKTPLQWINTEPDANTAAGACACCATTACAGTGGATTAACACTGAGCCAGACGCAAATCCTGATATGAAAGGPDMKGISSRGIWDMFMADCKKAGITATCTCTTCAAGAGGAATATGGGATATGTTTATGGCCGACTGCAAGAAAGCCGGTATKVFIDIHGIQPDSYTLPLWGDTEYLISTAAGGTGTTTATCGATATTCATGGTATCCAACCAGATTCTTATACATTGCCTTTATGGALEWFANEFKNDDTFIAIDVKNEPHGGAGATACAGAATACTTGATTTCCGCCTTAGAGTGGTTCGCAAACGAGTTCAAGAATQQGQGCGTGANDAVWESSTRSNNGACGATACTTTCATTGCCATCGACGTCAAGAACGAACCACATCAGCAAGGTCAGGGAWPYVAGLAGKRILAKNPGLLILVEGTGCGGTACTGGTGCAAATGACGCCGTGTGGGAATCTTCAACACGTTCTAACAATTGGNQCYKGDSSWWGGNLAGVKDIPVDCCTTATGTTGCGGGATTGGCGGGTAAAAGAATATTAGCTAAGAATCCAGGATTATTAVGNPKKLVYSPHEYGPSVNDQAWFATCTTGGTCGAAGGAAATCAATGCTACAAAGGTGATAGTTCCTGGTGGGGAGGTAACHPTINYDQLYSQHWHKHWLYIHEETTAGCTGGTGTCAAAGATATCCCTGTGGACGTTGGAAACCCAAAGAAGTTAGTGTATGIAPLLIGEWGGKLSGTNTQWMKLFTCCCCTCACGAATACGGTCCTTCTGTGAATGATCAAGCCTGGTTCCATCCAACTATTAVNLIAQYGLSHTFWCLNPNSGDTGGACTATGACCAGTTGTATTCCCAGCATTGGCACAAACATTGGTTGTATATCCACGAAGALLKDNWKDWDEEKYAFIKPCLGGSGGGTATTGCTCCATTATTGATAGGAGAATGGGGTGGAAAGTTATCCGGGACCAATAC LFKACAGTGGATGAAGTTATTCGTTAACTTAATCGCACAGTACGGTTTAAGTCACACTTTC(SEQ ID NO: 24)TGGTGCTTGAACCCAAACTCCGGAGATACCGGTGGATTGTTAAAGGATAATTGGAAAGACTGGGATGAGGAGAAATATGCTTTCATTAAGCCTTGTTTGGGTGGTTCCTTGTTTA AGTAAGGCGCGCC(SEQ ID NO: 4) NeotermesTTAATTAAAATGAGATTTCCATCTATTTTCACTGCTGTTTTGTTCGCAGCCTCAAGTGCAccession No: AB274614 koshunensisTTTAGCAGCCGATCCTGACTTGGTAAGATTACATGTCGATGGTAATAGAATCGTGATGMLGLLLSPSLSEADPDLVRLHVDGN symbiontGGAAAACCAGGTTTGGCTTCCTCTAAAACAGCTATGTTGAGAGGAGTGTCATGTAGTRIVMGKPGLASSKTAMLRGVSCSWTGGCACAACTGGTGGCCTCAATTTCATTCCGCCGCTACAGTTAGAGGTTTGAAATCTGHNWWPQFHSAATVRGLKSDFHANVACTTTCACGCAAATGTCGTGAGAACTTTCATAGGTGTTGAAAAGGAGGGAGGTTTCTVRTFIGVEKEGGFLTNQQKAYDCCYTAACAAACCAGCAAAAGGCTTATGATTGCTGTTACGCCGTAGTCGATGAATGCATCGAVVDECIAQGIYVIINWASFVLTYQTCACAAGGAATATACGTTATTATAAACTGGGCTTCATTCGTTTTGACCTACCAAACTCAQATQFFKTVATKYHSSSYVIYELLNAGCTACCCAGTTCTTCAAGACCGTTGCAACCAAATATCATAGTTCTTCTTACGTCATAEPEAATWAQIKPYSQALIQTIRAIDPSTACGAGTTATTGAACGAACCAGAAGCTGCGACATGGGCACAAATTAAACCTTATAGTNLILVPTPRWDQEIGAAANDPITGDNCAAGCTTTAATTCAAACAATCAGAGCTATTGACCCATCTAATTTGATATTAGTCCCAANLAYTLHIYTGTHPASYRDDARAAKCCCCTAGATGGGATCAAGAGATTGGTGCAGCTGCAAACGACCCTATCACAGGAGATAKKIPVWADENGAMNADGKGALDRACAATTTGGCTTATACTTTACACATATACACCGGAACACACCCAGCTAGTTATAGAGTGWNTWIAFYEELQIPWLGYGTQDTATGACGCCAGAGCAGCTAAGAAGAAAATCCCAGTGTGGGCCGACGAAAACGGTGCASETCSIFKSTDSFNDLSDWGKLLKETATGAACGCTGATGGAAAAGGAGCCTTGGATAGAACTGGTTGGAATACCTGGATCGCC IRKYQTTTTACGAAGAGTTACAGATACCTTGGTTGGGATATGGTACACAAGATACTTCCGAA(SEQ ID NO: 25)ACCTGTTCAATTTTCAAATCTACAGATTCCTTTAATGACTTGTCCGATTGGGGAAAGTTATTGAAGGAAACCATAAGAAAATACCAATAAGGCGCGCC (SEQ ID NO: 5) Hodoter-TTAATTAAAATGAGATTCCCTTCCATTTTCACTGCTGTTTTGTTCGCAGCCTCAAGTGCAccession No: AB274582 mopsisTTTAGCAGCCCCTCCATTTGGAAGATTATGCGTTGAAGGAAACAAAATCGTCGGTAAMLVLLLLHFINSKAPPFGRLCVEGN sjoestedtiCAAGAGAGCTCCTGGAGTGTTAAGAGGTGTTGGTTTGTCCTGGCATAATTGGTGGCCTKIVGNKRAPGVLRGVGLSWHNWW symbiontCAGTTTTACAACGCTGCAACCATCAATCACTTAAAGAACGACTTTCATGCCAATGTCAPQFYNAATINHLKNDFHANVIRAAITTAGAGCTGCTATAGGAGTGGAGAAAGAGAATGGTTACTTTGACAATCAGCAAAACGGVEKENGYFDNQQNAYDLLYAAVDCCTATGATTTGTTATACGCAGCTGTGGACGCAGCCTTGTCCGCTGGAATATATGTTATAALSAGIYVIVDWQAFQIHESDAKQCGTGGATTGGCAGGCCTTCCAAATCCACGAATCAGATGCAAAACAATTCTTTACTACFFTTVVNKYKGKSNVIYEIFNEPESAAGTTGTGAATAAGTACAAAGGTAAGAGTAACGTTATCTATGAGATATTTAATGAACCGWSEIKKYSISLIQTIRAIDSNAFILVPTGAATCCGCTGGTTGGTCTGAAATCAAGAAGTATTCAATTTCCTTAATTCAGACAATCTPNWDQYVEQAAADPISEYSNIAYTIAGAGCAATTGATTCCAACGCATTCATATTGGTTCCAACCCCTAATTGGGATCAGTATGHIYAATHPLSYLDNARTALKTIALFGTTGAACAGGCTGCAGCCGACCCTATTAGTGAGTACAGTAATATCGCCTATACAATTCTEIGAMEASGDGAIDQSKYQQWIDFACATATATGCCGCAACACATCCTTTATCTTATTTGGATAACGCTAGAACTGCCTTGAAYEQNGISYLCWAVQSKEETDSILKPSAACTATCGCCTTATTTGGGACCGAGATAGGTGCAATGGAGGCATCCGGTGATGGAGCEDWNDLTAWGKLCKSTITAHQAATAGACCAATCCAAGTACCAACAGTGGATCGATTTCTATGAGCAGAATGGAATCTC(SEQ ID NO: 26)ATACTTATGCTGGGCTGTACAGTCTAAAGAAGAGACTGACAGTATATTGAAACCAAGTGAAGATTGGAATGATTTGACAGCATGGGGAAAATTGTGTAAGTCAACAATTACTGCACACCAGTAAGGCGCGCC (SEQ ID NO: 6) ReticulitermesTTAATTAAAATGAGATTTCCATCTATTTTCACTGCTGTTTTGTTCGCAGCCTCAAGTGCAccession No: AB274534 speratusTTTAGCAGCCTTGGTACATATGGAATTAGAGAATAACTCCACTAGATTGAGAGTGAAMRKAMFVGLFLIALVHMELENNST symbiontGGGAAATAAGATCGTGGTCGGAAACTCTGATAAAGGTTTGAGATTAAGAGGAGTGARLRVKGNKIVVGNSDKGLRLRGVNATTTGTCCTGGAACAACTGGTGGCACCAATTCTACAACGCTGACACCGTTAGACACTTLSWNNWWHQFYNADTVRHLKNDFAAAGAACGACTTTCACGTCAATGTGATAAGAGCAGCCATTGGTGTGGAACAGGATGGHVNVIRAAIGVEQDGGWESNKQRSTGGATGGGAATCAAACAAGCAAAGAAGTTACGATGACTTGTACGCTGTTATCGACGCYDDLYAVIDACIANNVYVIVDWQTFATGTATCGCTAATAACGTCTATGTGATTGTCGATTGGCAGACTTTCTCTATCAAGTTGSIKLSEATEFFTNVANKYHSSSYIIYDTCAGAAGCCACAGAGTTCTTCACCAACGTTGCAAACAAATACCATAGTTCTTCCTATALLNEPDSSVPSWSAIKSYAESLIKTIRTCATCTACGACTTGTTGAACGAGCCTGATTCATCTGTGCCAAGTTGGTCCGCAATCAAAIDSSNLIIVPTPNWDQYVKQAAADGTCCTATGCCGAATCTTTGATAAAGACCATTAGAGCTATAGATTCCTCCAACTTAATAPITSDSNLIYSIHIYVGTHPMSYMDDATTGTGCCAACTCCAAATTGGGATCAGTACGTGAAGCAGGCTGCCGCAGATCCTATTAREALKTIPLIGGEIGAMNADGDGAACATCTGACAGTAACTTAATCTACTCAATACACATATACGTCGGTACTCACCCTATGA LDVSKFNQWIDFLGTTATATGGACGATGCTAGAGAAGCCTTAAAGACAATCCCTTTAATCGGAGGTGAAA(SEQ ID NO: 27)TAGGTGCAATGAATGCTGATGGTGACGGAGCTTTGGATGTTTCCAAATTCAACCAATGGATAGACTTCTTATAAGGCGCGCC (SEQ ID NO: 7) ReticulitermesTTAATTAAAATGAGATTTCCATCTATTTTCACAGCAGTTTTGTTCGCAGCCTCAAGTGAccession No: AB045179 speratusCTTTGGCCGGTGATTCCGGAAGAACAACCAGATATTGGGACTGTTGCAAAGCCTCTTMLLLFSLCLISWLVGDSGRTTRYWD symbiontGTGCTTGGGAAAAGAAAGCAGCCGTAACTCAACCTGTTGACACGTGCGGTAAGGACGCCKASCAWEKKAAVTQPVDTCGKDGAACCACTAGATTGGCTAGTAATGATACCGTGAAAAGTTCCTGTGACGGAGGTGATGGTTRLASNDTVKSSCDGGDGYMCYGATACATGTGTTATGATCAGGCACCATGGGCTGTTAACGATTCTGTAGCCTACGGTTTDQAPWAVNDSVAYGFAAAACCGGCGCCGCAGCTGCATGTTGTGGTGGAGAAACCGGTGCTTGCTGTAATTGCTATGAGTTGETGACCNCYELTFTSGPVNGKKMVACATTCACATCAGGTCCAGTGAATGGAAAAAAAATGGTGGTCCAGGTGACTAATACCVQVTNTGGDLGSNQFDLAIPGGGVGGGAGGTGATTTGGGAAGTAACCAGTTCGACTTAGCCATCCCAGGAGGTGGTGTCGGAIYNGCTQQSGAPADGWGSRYGGVSATATACAATGGTTGTACACAACAATCAGGTGCCCCTGCTGACGGTTGGGGATCAAGASRSECSQLPSGLQAGCQWRFDWFQTACGGAGGTGTCAGTTCTAGAAGTGAGTGTTCCCAGTTGCCATCAGGTTTACAAGCCNADNPSINFNQVTCPSELIARTNCKRGGATGCCAGTGGAGATTCGACTGGTTCCAAAACGCAGACAATCCTTCAATTAATTTC TAACCAAGTCACTTGTCCAAGTGAATTGATTGCAAGAACCAACTGCAAGAGAACATAA(SEQ ID NO: 28) GGCGCGCC (SEQ ID NO: 8) Pseudo-TTAATTAAAATGAGATTCCCTTCTATATTCACTGCTGTTTTGTTTGCAGCCAGTTCTGCAccession No: AB071864 trichonymphaCTTAGCACAGGCTGAGAATCACCCATCCTTGTCTTGGCAAAATTGTAGATCCGGTGGMFVFVLLWLTQSLGTGTNQAENHPS grassiiATCATGCACCCAAACCTCCGGTTCAGTTGTCTTGGATTCCAACATGAGATTTCCTTCTLSWQNCRSGGSCTQTSGSVVLDSNATCTTTACTGCTGTCTTATTCGCCGCTTCATCAGCTTTAGCATGGAGATGGACACACGWRWTHDSSLTNCYDGNEWSSSLCPATTCCAGTTTAACTAATTGTTATGATGGAAATGAGTGGAGTTCCTCATTATGCCCTGADPKTCSDNCLIDGADYSGTYGITSSGCCCTAAAACTTGTTCTGATAACTGTTTAATCGACGGTGCCGATTACTCTGGAACCTATNSLKLVFVTNGPYSTNIGSRVYLLKGGAATTACTTCCTCTGGAAACTCCTTGAAGTTGGTGTTCGTCACTAACGGACCTTACTDESHYQIFDLKNKEFTFTVDDSNLDCTACTAACATAGGTTCAAGAGTGTACTTGTTAAAAGACGAATCTCACTACCAAATATTCGLNGALYFVSMDEDGGTSRFSSNKTGACTTAAAGAACAAAGAGTTTACATTCACTGTTGATGATTCTAATTTGGACTGCGGAAGAKYGTGYCDAQCPHDIKFINGEATTAAACGGAGCCTTGTACTTTGTGAGTATGGATGAGGACGGTGGAACTTCAAGATTCNVENWKPQTNDENAGNGRYGACCTCTTCCAATAAGGCAGGAGCCAAATACGGTACTGGATATTGTGACGCCCAATGCCCATEMDIWEANKYATAYTPHICTVNGECACGATATTAAGTTCATTAACGGTGAAGCAAACGTTGAAAACTGGAAACCTCAAACCYRCDGSECGDTDSGNRYGGVCDKDAATGACGAAAATGCTGGTAACGGTAGATACGGAGCCTGCTGTACAGAGATGGATATAGCDFNSYRMGNTSFWGPGLIIDTGKTGGGAGGCAAATAAGTATGCTACTGCCTATACCCCTCACATCTGTACAGTCAACGGAPVTVVTQFVTKDGTDNGQLSEIRRKGAATATAGATGTGATGGTAGTGAATGTGGTGACACTGATTCCGGAAATAGATATGGAYVQGGKVIENTVVNIAGMSSGNSITGGAGTGTGCGATAAGGACGGATGCGATTTCAACTCTTATAGAATGGGTAACACTTCADDFCNEQKSAFGDTNDFEKKGGLSGTTTTGGGGTCCAGGATTGATCATTGACACAGGTAAGCCAGTTACTGTTGTAACCCAGTLGKAFDYGMVLVLSLWDDHQVNMTCGTAACCAAAGATGGAACTGACAACGGTCAATTGTCAGAGATAAGAAGAAAGTACLWLDSIYPTDQPASQPGVKRGPCATGTCCAGGGAGGTAAGGTTATTGAGAATACAGTAGTCAACATCGCGGGTATGTCCAGTSSGAPSDVESQHPDSSVTFSDIRFGPIGGTAATAGTATTACAGACGACTTTTGCAACGAGCAGAAATCAGCATTCGGAGACACT DSTYAACGATTTTGAAAAGAAGGGTGGATTATCCGGTTTGGGAAAAGCCTTCGATTATGGT(SEQ ID NO: 29)ATGGTGTTAGTTTTGTCTTTATGGGATGATCATCAAGTTAATATGTTATGGTTAGATTCCATATACCCTACCGACCAGCCTGCATCCCAGCCAGGTGTAAAGAGAGGACCATGTGCTACTTCATCTGGTGCCCCAAGTGATGTCGAATCACAACACCCTGACAGTTCCGTGACCTTTAGTGATATCAGATTTGGTCCAATAGATTCAACATATTAAGGCGCGCC (SEQ ID NO: 9)ReticulitermesTTAATTAAAATGAGATTCCCTTCCATTTTCACTGCCGTCTTATTTGCAGCCTCATCAGCAccession No: DQ014511 flavipes gutATTAGCCGAGAAACATCCTGCCTTTCAATGGAAGAAAGATGGTGTCACTCAAAATGGMLTVLFLLSLGWCEKHPAFQWKKD symbiontATTCTTGGTTCATGACAGACATGTGGGTGATAACTGGTATAGAGATCAGAAAGATGGGVTQNGFLVHDRHVGDNWYRDQKAAAATCCGGTGCTTTAGACTTAGACTACGAGAATGATGTTGGTGTTACTGTGTCCGGTDGKSGALDLDYENDVGVTVSGGTLGGTACTTTAACCCAGAGATTGGTGTCAAACTATAGTTGGAATAACAAGACCGTTGTATQRLVSNYSWNNKTVVGSRLYIMTGGGTCCAGATTATACATCATGACCGCCGACGAAAAGAAGTATGAGAAATTTAACTTAADEKKYEKFNLTGKEFTFTVNLAQIACTGGTAAGGAGTTTACCTTCACCGTCAATTTGGCCCAAATACCATGTGGTGTGAACGPCGVNAALYTVEMPADGIDATDQTCTGCATTATACACAGTGGAAATGCCTGCTGACGGAATTGACGCCACTGACCAAACCCQGAPYGYGYCDANCVDGGCCPEFDAGGGTGCACCATACGGTTACGGATATTGCGATGCAAACTGTGTTGATGGAGGTTGTTGIEATSKALVFTTHTCSGTGSGRGGGTCCTGAGTTTGATGGTATTGAAGCCACGAGTAAAGCATTAGTATTCACTACCCACACYTGCDTSGCGYNPYRDDNNHSFWTGTGCTCAGGTACTGGAAGTGGTAGAGGAGGTTACACCGGTTGTGATACATCCGGATGSSVNLAQPVTIVTQFQTNGDVTRKYITGGTTACAACCCTTATCGTGACGACAACAACCATTCTTTCTGGACCAGTTCAGTGAACQNGNPIDGGTLNQSRCSGKQNMTSTTTAGCTCAACCTGTGACTATAGTGACACAGTTCCAAACTAATGGTGATGTTACCAGAFSRGHVVVFSLWDSDGMSWLDGGNAAATATATTCAAAATGGAAACCCAATCGACGGTGGAACCTTAAACCAGAGTAGATGTAGPCTSYNIKDVETRTPNLTVTWSDTCCGGAAAGCAAAACATGACTTCTACCTTCTCTAGAGGTCATGTCGTGGTTTTCAGTT VKFGNIGSTTNTGTGGGATTCCGACGGAATGTCATGGTTAGATGGTGGTAATGCTGGACCTTGTACTTC(SEQ ID NO: 30)TTACAATATTAAAGATGTGGAAACAAGAACCCCAAACTTGACTGTAACCTGGTCCGATGTGAAATTCGGAAACATTGGATCAACAACTAATTAAGGCGCGCC (SEQ ID NO: 10)ReticulitermesTTAATTAAAATGAGATTTCCATCTATTTTCACTGCTGTTTTGTTCGCAGCCTCAAGTGCAccession No: DQ014512 flavipes gutTTTAGCACAATGGATGCAGATCGGTGGTAAGCAGAAATATCCTGCCTTTAAGCCAGGMVLCILLQWMQIGGKQKYPAFKPG symbiontTGCTAAGTACGGAAGAGGTTATTGTGACGGACAGTGCCCTCACGACATGAAGGTGTCAKYGRGYCDGQCPHDMKVSSGRATAGTGGAAGAGCAAACGTTGACGGATGGAAGCCACAAGACAACGACGAAAATAGTGNVDGWKPQDNDENSGNGKLGTCCGAAATGGAAAATTGGGTACATGTTGCTGGGAGATGGATATATGGGAAGGAAACTTAWEMDIWEGNLVSQAYTVHAGSKSGGTGTCCCAAGCCTACACCGTTCACGCTGGTTCCAAGTCCGGACAATATGAGTGTACTGQYECTGTQCGDTDSGERFKGTCDKGAACACAATGCGGTGACACCGACAGTGGTGAAAGATTCAAGGGAACATGCGATAAADGCDFASYRWGATDYYGPGKTVDTGATGGTTGTGATTTCGCAAGTTACAGATGGGGAGCTACAGACTATTACGGTCCTGGAKQPMTVVTQFIGDPLTEIKRVYVQGAAGACCGTGGACACCAAACAGCCAATGACAGTCGTGACCCAGTTCATTGGTGACCCTGKVINNSKTSNLGSVYDSLTEAFCDTTGACTGAGATAAAGAGAGTTTATGTACAAGGAGGAAAAGTCATAAACAATTCCAAADTKQVTGDTNDFKAKGGMSGFSKNACATCTAACTTAGGTTCAGTGTACGATTCTTTGACTGAGGCCTTCTGCGATGACACCALDTPQVLVMSLWDDHTANMLWLDAACAGGTTACAGGTGATACAAATGACTTTAAGGCTAAAGGAGGTATGTCTGGATTCTSTYPTDSTKPGAARGTCAVTSGDPKCCAAGAACTTAGACACCCCACAAGTTTTGGTGATGTCTTTATGGGATGACCATACAGDVESKQANSQVVYSDIKFGPINSTYCTAATATGTTATGGTTAGATTCTACTTATCCTACCGATAGTACAAAGCCAGGTGCCGC KANAAGAGGTACTTGTGCCGTCACCTCCGGGGACCCTAAAGATGTGGAATCCAAGCAAGC(SEQ ID NO: 31)CAACTCTCAGGTAGTTTACAGTGACATTAAGTTTGGTCCTATTAATTCAACATACAAAGCAAATTAAGGCGCGCC (SEQ ID NO: 11) ReticulitermesTTAATTAAAATGAGATTTCCATCTATTTTCACTGCTGTTTTGTTCGCAGCCTCCAGTGCAccession No: DQ014513 flavipes gutATTAGCAGAGTTTACATTCACAACCGATGTATCCGGTTTACCTTGTGGGTTAAACGGTMFKLKNKEFTFTTDVSGLPCGLNGA symbiontGCCTTGTACTTTGTCGCCATGGACGAGGACGGAGGTAAAGCAAAGCATCCATTATCCLYFVAMDEDGGKAKHPLSKPGAKYAAACCAGGTGCTAAGTACGGAATGGGTTACTGTGACGCCCAATGTCCACACGATATGGMGYCDAQCPHDMKFIEGLANCEGAAGTTTATCGAAGGATTGGCAAACTGCGAGGGTTGGAAGCCTCAGGATAATGACGAAWKPQDNDENSGNGKYGTCCAEMDIAACTCAGGTAATGGAAAATACGGTACTTGTTGCGCTGAAATGGATATATGGGAGGCCWEANSQATAYTVHACSKTGATKWSAACAGTCAAGCAACAGCTTATACTGTGCATGCCTGTTCCAAGACCGGAGCAACCAAAGNDCGDDDNRYNGICDKDGCDYNSTGGTCCGGAAATGACTGTGGTGATGATGACAACAGATACAATGGAATTTGCGATAAGWRLGNQTFFGPGLIVDSSKPVTVVTGACGGTTGCGATTACAACTCATGGAGATTAGGTAATCAGACTTTCTTCGGACCTGGTTQFITSNNQDSGELVEVRRLYVQNNKTAATTGTAGATAGTTCCAAACCTGTAACAGTCGTGACCCAATTCATAACTTCCAATAAVIQNTVTNIQGIKNADSITDSFCDDTCCAAGATTCAGGAGAATTAGTCGAGGTTAGAAGATTGTACGTCCAGAACAACAAAGTKSVFGDTNDYKAKGAMAGFSKSIDPCATCCAGAACACTGTTACTAACATCCAGGGTATAAAGAATGCTGATTCTATTACCGATGVVLVRSLWDDHSVNMLWLDSTYPTCCTTTTGCGATGATACAAAGTCCGTTTTCGGTGACACTAATGACTATAAGGCCAAGGTDSNKPGASRGPCAITSGKPSDVESQGAGCAATGGCTGGATTTTCAAAGAGTATCGATCCAGGTGTAGTCTTAGTGAGAAGTTSASASVKFSDIRFGPIDSTYSKTGTGGGACGATCACTCCGTTAATATGTTATGGTTGGATTCAACCTACCCTACAGACAG(SEQ ID NO: 32)TAACAAACCAGGAGCCAGTAGAGGTCCTTGCGCAATTACTTCAGGAAAACCATCTGATGTAGAATCCCAGTCCGCTTCTGCATCTGTCAAGTTCTCCGATATTAGATTCGGTCCAATAGATTCTACTTATAGTAAATAAGGCGCGCC (SEQ ID NO: 12) MastotermesTTAATTAAAATGAGATTCCCAAGTATATTTACTGCTGTTTTGTTCGCAGCCAGTTCTGAccession No: AJ511343 darwinensisCTTTAGCAGCCTATGATTACAATGACGTATTAACCAAAAGTTTGTTGTTCTACGAAGCMRVLLCLLSAFALCQGAYDYNDVLTCAAAGATCCGGTAAGTTACCTTCTGATCAGAAAGTCACCTGGAGAAAAGATTCAGCTKSLLFYEAQRSGKLPSDQKVTWRKATTAAACGATAAGGGACAAAATGGTGAGGACTTAACTGGTGGATATTATGACGCCGGDSALNDKGQNGEDLTGGYYDAGDYTGATTACGTGAAGTTTGGTTTTCCAATGGCATATACTGCTACCGTTTTGGCTTGGGGTVKFGFPMAYTATVLAWGLVDHPAGTTAGTGGACCATCCTGCCGGATACAGTTCTGCGGGTGTCTTGGATGATGGTAGAAAAYSSAGVLDDGRKAVKWVTDYLIKAGCTGTGAAGTGGGTTACCGATTACTTAATCAAAGCCCACGTATCAAAGAACGAATTAHVSKNELYGQVGDGDADHAYWGRTACGGACAGGTCGGTGACGGTGACGCAGATCACGCTTATTGGGGACGTCCAGAGGATPEDMTMARPAYKIDTSRPGSDLAGEATGACAATGGCAAGACCAGCATACAAAATAGACACTTCAAGACCAGGTTCCGACTTATAAALAAASIVFKSTDSNYANTLLTGCGGGTGAAACCGCAGCGGCATTGGCTGCTGCATCTATTGTGTTTAAGTCAACAGATHAKQLFDFANNYRGKYSDSITQASNTCTAATTACGCCAACACCTTATTGACCCACGCAAAACAATTATTCGACTTTGCCAATAFYSSSDYKDELVWAAVWLYRATNDACTATAGAGGTAAGTATAGTGATTCCATAACACAGGCATCTAATTTCTACAGTAGTTCQTYLTTAEKLYSDLGLQSWNGGFTCGACTATAAAGATGAATTGGTTTGGGCAGCTGTATGGTTGTACAGAGCCACTAACGAWDTKISGVEVLLAKITGKQAYKDKTCAGACCTATTTGACAACTGCAGAGAAGTTATACTCAGACTTGGGATTACAGTCCTGVKGYCDYISGSQQKTPKGLVYIDKWGAACGGAGGTTTCACATGGGACACCAAAATTAGTGGAGTAGAAGTGTTATTGGCTAAGSLRMAANAAYICAVAADVGISSTAGATTACTGGTAAACAGGCATATAAGGACAAAGTAAAGGGATATTGTGATTATATCTCYRQFAKTQlNYILGDAGRSFVVGYGAGGATCTCAGCAGAAAACACCTAAAGGATTAGTTTACATAGATAAGTGGGGTTCCTTNNPPTHPHHRSSSCPDAPATCDWNNAAGAATGGCCGCAAACGCCGCATATATTTGCGCTGTAGCCGCAGACGTCGGAATCAGYNSANPNPHVLYGALVGGPDSNDNTTCAACAGCTTACAGACAGTTCGCCAAAACACAGATTAATTACATATTGGGTGATGCYQDLRSDYVANEVATDYNAAFQSLCGGACGTTCTTTTGTGGTTGGTTACGGAAACAACCCACCTACACACCCACATCACAG LALIVDLGLATCCAGTTCATGTCCTGACGCCCCAGCAACATGCGATTGGAATAACTACAACAGTGC(SEQ ID NO: 33)TAACCCTAATCCACATGTTTTATACGGTGCATTAGTTGGTGGACCAGATTCCAACGATAATTATCAAGACTTAAGATCAGATTATGTCGCCAACGAAGTGGCAACAGACTACAATGCAGCCTTCCAGTCATTGTTAGCATTAATCGTGGACTTAGGTTTGTAAGGCGCGCC (SEQ ID NO: 13)ReticulitermesTTAATTAAAATGAGATTTCCAAGTATATTTACTGCCGTCTTATTTGCAGCCTCAAGTGAccession No: AY572862 flavipesCTTTAGCCGCTTATGACTACAAAACAGTATTGTCCAATTCCTTGTTGTTCTACGAAGCMKVFVCLLSALALCQAAYDYKTVLTCAAAGATCCGGTAAGTTACCTTCTGATCAGAAAGTCACTTGGAGAAAGGATTCAGCSNSLLFYEAQRSGKLPSDQKVTWRKATTAAACGACAAAGGACAAAAGGGTGAGGACTTGACTGGAATGAGATTCCCATCAADSALNDKGQKGEDLTGGYYDAGDFTATTCACCGCCGTGTTGTTTGCTGCATCTTCAGCTTTAGCCGGTTATTACGATGCCGGTVKFGFPMAYTVTVLAWGVIDYESAGATTTCGTCAAATTCGGATTTCCAATGGCTTACACTGTAACTGTCTTGGCCTGGGGTGYSAAGALDSGRKALKYGTDYFLKATTATTGATTACGAAAGTGCATACTCTGCTGCCGGAGCATTGGATTCAGGTAGAAAGGHTAANEFYGQVGQGDVDHAYWGRCATTAAAGTACGGGACCGACTATTTCTTAAAGGCCCATACAGCTGCCAATGAGTTCTPEDMTMSRPAYKIDTSKPGSDLAAEATGGACAGGTAGGTCAAGGAGATGTGGACCATGCATATTGGGGACGTCCAGAGGATTAAALAATAIAYKSADATYSNNLITATGACTATGTCTCGTCCTGCTTACAAAATAGACACCTCCAAGCCAGGTTCCGACTTAGHAKQLFDFANNYRGKYSDSITDAKNCTGCAGAGACTGCAGCTGCCTTAGCCGCAACAGCCATCGCATACAAATCAGCTGATGFYASGDYKDELVWAAAWLYRATNCAACATATTCCAATAACTTGATAACTCATGCAAAGCAGTTATTCGACTTTGCTAACAADNTYLTKAESLYNEFGLGNWNGAFCTATAGAGGAAAATATAGTGATTCCATTACCGATGCCAAGAACTTTTATGCCTCAGGNWDNKISGVQVLLAKLTSKQAYKDAGATTATAAAGACGAATTAGTCTGGGCCGCTGCATGGTTATACAGAGCTACAAATGAKVQGYVDYLISSQKKTPKGLVYIDQCAACACATATTTGACCAAGGCTGAATCCTTATACAATGAGTTCGGATTGGGAAACTGWGTLRHAANSALIALQAADLGINAAGAATGGTGCCTTCAATTGGGATAACAAAATCAGTGGAGTACAGGTCTTATTGGCCAATYRAYAKKQIDYALGDGGRSYVVGGTTAACATCAAAACAGGCATACAAGGATAAGGTTCAGGGTTACGTGGATTACTTGATFGTNPPVRPHHRSSSCPDAPAVCDWCTCCTCCCAAAAAAAGACCCCTAAGGGATTAGTTTACATTGATCAATGGGGAACCTTNTYNSAGPNAHVLTGALVGGPDSNGAGACACGCTGCTAATAGTGCCTTAATCGCGTTGCAGGCTGCCGACTTAGGTATTAADSYTDARSDYISNEVATDYNAGFQSCGCAGCTACCTATAGAGCCTACGCAAAGAAGCAAATCGACTATGCTTTGGGTGATGG AVAGLLKAGVTGGACGTTCTTATGTGGTGGGTTTTGGTACTAACCCACCTGTAAGACCACATCACAGA(SEQ ID NO: 34)AGTTCCAGTTGTCCTGACGCCCCAGCAGTCTGCGATTGGAACACCTACAATTCAGCTGGTCCAAACGCCCACGTGTTAACTGGTGCCTTAGTTGGTGGACCTGATTCTAATGATTCCTATACTGATGCTAGATCAGACTACATTTCTAACGAGGTTGCAACTGATTACAACGCCGGATTTCAGAGTGCTGTCGCTGGATTATTAAAGGCTGGAGTGTAAGGCGCGCC (SEQ ID NO: 14)ReticulitermesTTAATTAAAATGAGATTCCCAAGTATATTTACTGCCGTCTTATTTGCAGCCTCCAGTGAccession No: AB019095 speratusCATTAGCCGCTTATGACTACAAAACAGTATTGTCCAATTCCTTGTTGTTCTACGAAGCMKVFVCLLSALALCQAAYDYKTVLTCAAAGATCCGGTAAGTTACCTTCTGACCAGAAAGTGACCTGGAGAAAGGATTCAGCSNSLLFYEAQRSGKLPSDQKVTWRKATTAAACGACAAAGGACAAAAGGGTGAGGACTTAACCGGTGGATATTACGACGCCGDSALNDKGQKGEDLTGGYYDAGDFGAGACTTTGTGAAATTTGGTTTTCCAATGGCTTACACAGTTACCGTATTGGCATGGGGVKFGFPMAYTVTVLAWGVIDYESATGTTATTGATTACGAATCCGCCTACTCTGCCGCAGGAGCTTTAGATTCAGGTAGAAAGYSAAGALDSGRKALKYGTDYFLKAGCCTTGAAATATGGGACCGACTATTTCTTAAAGGCACATACAGCAGCTAACGAGTTTHTAANEFYGQVGQGDVDHAYWGRTACGGACAGGTGGGTCAAGGTGACGTTGACCACGCATACTGGGGACGTCCTGAAGATPEDMTMSRPAYKIDTSKPGSDLAAEATGACCATGAGCAGACCAGCATACAAAATAGACACTTCTAAGCCTGGTTCCGACTTATAAALAATAIAYKSADATYSNNLITGCTGCAGAGACTGCAGCTGCATTAGCAGCCACAGCTATTGCATACAAATCTGCCGATHAKQLFDFANNYRGKYSDSITDAKNGCAACATATTCCAACAATTTGATAACACATGCAAAACAATTATTCGACTTTGCCAACFYASGDYKDELVWAAAWLYRATNAATTACAGAGGAAAATATTCCGATAGTATTACCGATGCCAAGAACTTTTATGCTTCTGDNTYLTKAESLYNEFGLGNFNGAFNGTGATTACAAAGACGAATTGGTATGGGCCGCTGCATGGTTGTACAGAGCAACCAATGWDNKVSGVQVLLAKLTSKQVYKDACAACACATATTTGACTAAGGCAGAATCCTTATACAATGAATTTGGTTTGGGAAACTTKVQSYVDYLISSQKKTPKGLVYIDQCAATGGTGCCTTCAATTGGGATAACAAAGTCTCCGGAGTCCAGGTGTTATTGGCCAAWGTLRHAANSALIALQAADLGINAAGTTAACCTCAAAACAAGTGTATAAGGATAAGGTACAGTCTTACGTGGACTATTTGATTYRAYAKKQIDYALGDGGRSYVIGFCTCCTCACAAAAAAAGACACCAAAAGGTTTAGTGTACATCGATCAATGGGGTACTTTGTNPPVRPHHRSSSCPDAPAVCDWNAAGACACGCAGCTAATTCTGCTTTGATCGCTTTGCAGGCAGCTGACTTAGGAATTAACTYNSAGPNAHVLTGALVGGPDSNDGCTGCTACTTACAGAGCCTACGCAAAGAAGCAAATCGACTATGCTTTGGGTGATGGTSYTDARSDYISNEVATDYNAGFQSAGGAAGATCCTATGTTATTGGATTTGGGACCAACCCTCCAGTAAGACCACATCACAGA VAGLLKAGVAGTTCATCTTGCCCAGATGCACCAGCTGTCTGCGATTGGAACACCTATAACTCCGCTG(SEQ ID NO: 35)GTCCAAACGCCCACGTGTTAACCGGTGCATTGGTTGGAGGACCTGATAGTAATGATAGTTATACCGATGCTCGTTCTGACTACATATCCAACGAAGTGGCAACTGATTACAATGCGGGTTTCCAATCCGCTGTCGCTGGATTATTGAAGGCGGGTGTCTAAGGCGCGCC (SEQ ID NO: 15)Coptotermes TTAATTAAAATGAGATTCCCTTCCATTTTCACTGCTGTTTTGTTCGCAGCCTCAAGTGCAccession No: AB058671 formosanusTTTAGCAGCCTATGACTACAAGACAGTATTGAAGAACTCCTTGTTGTTCTACGAAGCTMRVFVCLLSALALCQAAYDYKTVLCAAAGAAGTGGAAAATTGCCTGCAGACCAGAAGGTGACCTGGAGAAAAGATTCCGCKNSLLFYEAQRSGKLPADQKVTWRATTAAACGACAAGGGACAGAAGGGAGAGGACTTAACTGGAGGTTATTACGACGCCGKDSALNDKGQKGEDLTGGYYDAGDGAGACTTTGTGAAGTTCGGTTTTCCAATGGCATACACAGTTACCGTGTTGGCCTGGGGFVKFGFPMAYTVTVLAWGLVDYESTTTAGTCGATTATGAATCTGCTTACAGTACTGCGGGTGCCTTGGATGATGGTAGAAAGAYSTAGALDDGRKALKWGTDYFLKGCCTTGAAATGGGGTACAGATTATTTCTTGAAAGCACATACCGCTGCCAATGAGTTTTAHTAANEFYGQVGQGDVDHAYWGACGGACAGGTGGGTCAGGGAGATGTGGATCATGCTTACTGGGGACGTCCTGAGGACARPEDMTMSRPAYKIDTSKPGSDLAATGACTATGTCTAGACCAGCTTACAAGATCGATACATCAAAACCTGGTAGTGACTTAGETAAALAATAIAYKSADSTYSNNLITCTGCAGAAACAGCAGCCGCTTTAGCAGCAACCGCAATAGCTTACAAGTCAGCCGATTHAKQLFDFANNYRGKYSDSITDAKNCTACCTACAGTAACAACTTAATTACTCATGCAAAGCAGTTGTTCGATTTTGCAAACAAFYASGDYKDELVWAAAWLYRATNTTATAGAGGAAAGTACTCTGATAGTATTACCGATGCCAAGAATTTCTATGCATCCGGTDNTYLTKAESLYNEFGLGSWNGAFGATTATAAGGACGAATTAGTATGGGCTGCAGCCTGGTTGTATAGAGCTACAAATGATNWDNKISGVQVLLAKLTSKQAYKDAACACTTACTTAACCAAAGCCGAATCATTGTATAATGAATTTGGTTTAGGATCTTGGAKVQGYVDYLVSSQKKTPKGLVYIDACGGTGCATTCAATTGGGATAACAAGATATCCGGAGTTCAGGTCTTATTAGCCAAATQWGTLRHAANSALIALQAADLGINATGACATCCAAACAAGCATACAAAGATAAAGTTCAGGGTTATGTTGATTACTTAGTCTASYRQYAKKQIDYALGDGGRSYVVCCTCTCAAAAGAAAACTCCAAAGGGATTGGTCTATATTGACCAATGGGGAACCTTAAGFGTNPPVRPHHRSSSCPDAPAACDGACACGCAGCTAATAGTGCCTTGATCGCTTTACAGGCCGCTGATTTGGGTATAAACGWNTYNSAGPNAHVLTGALVGGPDSCTGCTAGTTATAGACAATACGCAAAGAAGCAAATTGATTATGCCTTAGGTGACGGAGNDSYTDSRSDYISNEVATDYNAGFQGTCGTTCTTACGTGGTCGGATTCGGAACTAACCCTCCAGTAAGACCTCATCATAGATC SAVAGLLKAGVCAGTTCCTGTCCTGACGCACCAGCCGCTTGCGACTGGAATACTTACAACTCTGCCGGA(SEQ ID NO: 36)CCAAATGCCCACGTCTTGACCGGAGCCTTAGTAGGTGGACCAGATTCCAACGATAGTTACACAGATTCACGTTCTGATTATATCAGTAACGAAGTCGCTACTGATTACAATGCCGGTTTCCAATCTGCAGTTGCTGGTTTGTTGAAAGCCGGAGTATAAGGCGCGCC (SEQ ID NO: 16)Coptotermes TTAATTAAAATGAGATTCCCTAGTATTTTCACTGCCGTCTTATTTGCAGCCAGTTCTGCAccession No: AF336120 acinaciformisTTTAGCCGCATATGATTATACCACAGTTTTGAAAAGTTCCTTATTGTTCTACGAAGCTMRVFVCLLSALALCQAAYDYTTVLCAAAGATCCGGTAAGTTGCCAGCCGACCAGAAGGTCACTTGGAGAAAAGATTCAGCKSSLLFYEAQRSGKLPADQKVTWRATTAGACGATAAAGGAAATAATGGAGAGGACTTAACAGGAGGTTATTATGACGCTGKDSALDDKGNNGEDLTGGYYDAGDGTGATTTTGTGAAGTTTGGTTTTCCTTTAGCATACACCGCTACTGTTTTAGCCTGGGGTFVKFGFPLAYTATVLAWGLVDYEATTGGTGGACTATGAAGCGGGTTACTCATCCGCTGGAGCCACAGATGACGGTAGAAAGGYSSAGATDDGRKAVKWATDYLLKGCAGTGAAATGGGCAACCGACTATTTGTTGAAGGCACATACTGCCGCTACCGAGTTAAHTAATELYGQVGDGDADHAYWGTACGGACAGGTCGGGGACGGTGACGCCGATCACGCATATTGGGGACGTCCTGAAGATRPEDMTMARPAYKIDASRPGSDLAGATGACTATGGCTAGACCAGCATACAAGATCGACGCTAGCAGACCAGGATCTGACTTAETAAALAAASIVFKGVDSSYSDNLLGCGGGTGAAACCGCTGCCGCTTTAGCCGCTGCATCCATAGTTTTCAAAGGTGTAGATTAHAKQLFDFADNYRGKYSDSITQASCTTCATATTCTGACAACTTGTTAGCTCACGCTAAACAGTTATTTGATTTCGCTGACAANFYASGDYKDELVWAATWLYRATTTATAGAGGAAAATACAGTGATTCCATAACACAAGCTTCAAACTTTTACGCCTCCGGNDNTYLTKAESLYNEFGLGNWNGAAGATTACAAAGACGAGTTAGTCTGGGCTGCCACTTGGTTGTACAGAGCAACCAACGAFNWDNKVSGVQVLLAKLTSKQAYKTAATACATATTTGACCAAAGCAGAATCCTTGTACAACGAGTTCGGATTAGGAAACTGDTVQGYVDYLINNQQKTPKGLLYIDGAACGGAGCCTTTAATTGGGACAACAAGGTGTCCGGTGTTCAGGTGTTGTTAGCCAAQWGTLRHAANAALIILQAADLGISAATTGACCTCCAAGCAGGCTTATAAAGACACCGTTCAAGGATACGTCGATTATTTGATTDSYRQFAKKQIDYALGDGGRSYVVAACAATCAGCAAAAGACCCCAAAGGGTTTGTTATACATAGACCAATGGGGGACCTTGGFGDNPPTHPHHRSSSCPDAPAVCDAGACACGCAGCTAATGCTGCCTTAATAATCTTACAGGCTGCTGATTTGGGTATTTCTGWNTFNSPDPNFHVLTGALVGGPDQCCGACAGTTATAGACAATTCGCAAAGAAGCAAATAGATTACGCTTTAGGTGACGGAGNDNYVDDRSDYVSNEVATDYNAGFGTAGATCATATGTAGTTGGTTTTGGAGACAATCCTCCAACACATCCTCATCACCGTTC QSAVAALVTLGVTTCCTCATGCCCTGACGCCCCAGCAGTATGCGATTGGAATACTTTCAATTCACCTGAT(SEQ ID NO: 37)CCAAACTTTCATGTCTTAACCGGAGCTTTAGTGGGAGGTCCTGATCAGAACGATAACTACGTTGATGATCGTTCTGACTACGTGTCCAACGAGGTTGCAACCGACTATAATGCAGGATTCCAAAGTGCTGTGGCCGCTTTAGTTACTTTAGGAGTTTAAGGCGCGCC (SEQ ID NO: 17)Nasutitermes TTAATTAAAATGAGATTTCCATCTATTTTCACTGCCGTCTTATTTGCAGCCTCAAGTGCAccession No: AB013273 walkeriTTTAGCAGCCTATGATTACAAACAAGTATTGAGAGATTCCTTATTGTTCTACGAAGCTMRVFLCLLSALALCQAAYDYKQVLCAGAGAAGCGGTAGATTACCAGCAGACCAGAAGGTCACCTGGAGAAAAGATTCCGCRDSLLFYEAQRSGRLPADQKVTWRCTTGAATGATCAGGGAGAGCAAGGTCAAGACTTAACCGGAGGTTATTTTGACGCCGGKDSALNDQGEQGQDLTGGYFDAGDTGATTTTGTGAAGTTTGGATTCCCAATGGCTTATACAGCAACCGTTTTGGCCTGGGGTFVKFGFPMAYTATVLAWGLIDFEAGTTAATCGACTTTGAAGCCGGTTACTCTTCTGCTGGTGCCTTGGACGATGGTAGAAAAGYSSAGALDDGRKAVKWATDYFIKACAGTAAAGTGGGCTACTGATTACTTTATAAAAGCCCATACTTCTCAAAACGAGTTTTAHTSQNEFYGQVGQGDVDHAYWGRCGGACAAGTCGGTCAGGGTGACGTAGATCACGCATATTGGGGACGTCCTGAAGATATPEDMTMARPAYKIDTSRPGSDLAGEGACAATGGCTAGACCAGCCTACAAGATTGATACCAGCAGACCAGGTAGTGACTTAGCTAAALAAASIVFKNVDGTYSNNLLTAGGAGAAACTGCTGCAGCTTTGGCTGCCGCATCCATCGTTTTCAAGAATGTAGATGGHARQLFDFANNYRGKYSDSITDARNTACATATTCCAACAACTTACTTACTCATGCTAGACAGTTGTTTGATTTCGCCAACAATFYASADYRDELVWAAAWLYRATNTACAGAGGAAAATACTCTGATAGTATTACCGATGCAAGAAACTTTTACGCTAGTGCCDNSYLNTAESLYNEFGLQNWGGGLGACTATAGAGATGAGTTAGTCTGGGCAGCTGCCTGGTTGTACAGAGCAACCAACGACNWDSKVSGVQVLLAKLTNKQEYKDAATTCTTACTTGAACACTGCTGAATCATTATACAACGAGTTTGGATTGCAAAATTGGGTIQSYVNYLINNQQKTPKGLLYIDMGTGGAGGGTTAAACTGGGATTCTAAAGTGAGTGGTGTTCAAGTTTTGTTAGCCAAGTTWGTLRHAANAAFIMLEAADLGLSAGACCAACAAACAAGAGTATAAGGACACTATTCAATCATACGTGAATTACTTAATCAASSYRQFAQTQIDYALGDGGRSFVCGTAACCAACAGAAAACTCCAAAGGGATTGTTATACATTGACATGTGGGGGACCTTGAGFGSNPPTRPHHRSSSCPPAPATCDWNACACGCAGCTAACGCAGCCTTTATAATGTTAGAAGCTGCCGACTTAGGTTTATCCGCTTFNSPDPNYNVLSGALVGGPDQNDNTCATCTTATAGACAGTTCGCCCAAACACAAATAGACTACGCATTGGGGGACGGTGGAYVDDRSDYVHNEVATDYNAGFQSACGTTCTTTTGTCTGTGGTTTCGGTTCTAATCCTCCAACTAGACCTCATCATAGATCCAG LAALVALGYTTCATGCCCGCCTGCTCCAGCTACCTGTGATTGGAATACATTCAATTCTCCTGACCCA(SEQ ID NO: 38)AACTACAATGTTTTATCCGGTGCCTTGGTTGGTGGTCCTGACCAGAATGATAACTACGTGGACGATAGAAGTGATTATGTCCATAATGAGGTAGCAACTGACTACAATGCCGGTTTCCAATCAGCCTTAGCCGCTTTAGTCGCCTTAGGTTACTAAGGCGCGCC (SEQ ID NO: 18)Nasutitermes TTAATTAAAATGAGATTTCCATCTATTTTCACTGCCGTCTTATTTGCAGCCTCCAGTGCAccession No: AB013272 takasagoensisATTAGCAGCCTATGATTATAAACAAGTTTTGAGAGATTCCTTATTGTTCTACGAAGCTMRVFLCLLSALALCQAAYDYKQVLCAGAGAAGCGGTAGATTACCAGCAGACCAGAAGGTCACTTGGAGAAAAGATTCAGCRDSLLFYEAQRSGRLPADQKVTWRCTTGAATGATCAGGGAGATCAAGGTCAAGACTTAACCGGAGGTTATTTTGACGCCGGKDSALNDQGDQGQDLTGGYFDAGDTGATTTTGTGAAATTTGGTTTCCCAATGGCATATACTGCTACCGTCTTGGCCTGGGGTFVKFGFPMAYTATVLAWGLIDFEAGTTAATCGATTTTGAGGCAGGATACAGTTCCGCTGGTGCCTTGGATGACGGTAGAAAAYSSAGALDDGRKAVKWATDYFIKAGCAGTAAAGTGGGCAACTGATTACTTTATAAAGGCCCACACTTCACAGAATGAGTTTHTSQNEFYGQVGQGDADHAFWGRPTACGGACAAGTCGGTCAGGGTGACGCTGATCACGCTTTCTGGGGACGTCCTGAAGATEDMTMARPAYKIDTSRPGSDLAGETATGACCATGGCTAGACCAGCCTACAAGATTGACACCAGCAGACCAGGTAGTGACTTAAAALAAASIVFRNVDGTYSNNLLTHGCGGGTGAAACCGCAGCGGCATTGGCAGCTGCCAGTATCGTGTTTAGAAATGTTGATARQLFDFANNYRGKYSDSITDARNFGGTACATACTCTAACAACTTACTTACTCATGCCAGACAATTATTTGACTTTGCAAATAYASADYRDELVWAAAWLYRATNDACTACAGAGGAAAATACTCAGATTCCATAACCGACGCTAGAAACTTTTACGCCAGTGNTYLNTAESLYDEFGLQNWGGGLNCAGATTACCGTGACGAATTGGTTTGGGCTGCCGCATGGTTGTACAGAGCTACAAATGWDSKVSGVQVLLAKLTNKQAYKDTACAACACTTACTTGAATACCGCAGAATCCTTGTATGATGAATTTGGATTGCAGAACTGVQSYVNYLINNQQKTPKGLLYIDMGGGTGGAGGGTTAAACTGGGATTCAAAGGTGTCTGGTGTCCAGGTCTTGTTAGCAAAWGTLRHAANAAFIMLEAAELGLSASATTGACCAACAAACAGGCTTACAAAGATACTGTGCAGTCTTACGTGAATTACCTGATSYRQFAQTQIDYALGDGGRSFVCGFTAATAACCAGCAAAAGACCCCAAAAGGATTGTTATACATTGATATGTGGGGTACATTGSNPPTRPHHRSSSCPPAPATCDWNGAGACACGCCGCAAATGCTGCATTCATCATGTTGGAAGCTGCCGAGTTGGGTTTATCTFNSPDPNYHVLSGALVGGPDQNDNCGCATCATCTTACAGACAGTTTGCTCAAACTCAGATCGACTACGCTTTGGGTGACGGTYVDDRSDYVHNEVATDYNAGFQSAGGAAGAAGTTTCGTCTGTGGTTTTGGTTCAAACCCTCCTACAAGACCACATCATCGTT LAALVALGYCTTCCAGTTGCCCGCCTGCCCCAGCAACTTGTGACTGGAATACATTCAACTCACCTGA(SEQ ID NO: 39)CCCAAATTACCACGTGTTATCTGGAGCTTTGGTAGGAGGACCAGATCAAAACGATAATTATGTGGATGATAGATCCGACTACGTCCATAACGAAGTGGCAACCGACTACAACGCCGGATTTCAGAGTGCTTTGGCAGCCTTAGTTGCTTTGGGTTATTAAGGCGCGCC (SEQ ID NO: 19)Panesthia TTAATTAAAATGAGATTTCCATCTATTTTCACTGCTGTTTTGTTCGCAGCCTCAAGTGCAccession No: AF220597 cribrataTTTAGCCGCAACTTATGATTACTCCCAATTGATCCAGTATTCCTTATTGTTCTACGAGGMKIILLFLGGLALCQGATYDYSQLIQCTCAGAGAAGTGGAAAATTGCCAGCCGATCAGAAGGTGACCTGGAGAAAAGATTCCYSLLFYEAQRSGKLPADQKVTWRKGCATTAAATGACAAGGGACAAAATGGTGAGGACTTAACTGGAGGATATTACGATGCCDSALNDKGQNGEDLTGGYYDAGDYGGTGATTATGTCAAATTTGGATACCCAATGGCCTTTACAGCAACCTTGTTAGCCTGGAVKFGYPMAFTATLLAWSLIDYEQGYGTTTGATTGACTATGAACAAGGTTATGCAAAGGCTAATTCCGTCGAGGACGCGAGAAAKANSVEDARKAVKWATDYFLKAAGGCAGTGAAATGGGCCACTGACTATTTCTTAAAAGCCCATGTATCAGAACACGAGTHVSEHEFYGQVGEGNLDHNSWGRPTCTACGGACAGGTGGGAGAGGGAAACTTGGATCATAATTCATGGGGACGTCCTGAGGEDMTMERPAYKIDEQNPGTELAAETACATGACTATGGAAAGACCAGCATATAAGATTGATGAGCAAAACCCTGGAACCGAAAAALAAASIVFKSVDPSYSNTLLTHTTAGCTGCCGAAACTGCTGCAGCCTTAGCCGCTGCCTCCATCGTGTTCAAATCTGTTGAKQLYDFGDNFRGKYSESINDAQQFACCCTAGTTACTCCAATACATTACTTACTCACGCTAAACAATTGTATGACTTTGGTGAYRSNEFEDELVWGALWLYKATMDETAACTTTAGAGGAAAATACAGTGAATCCATAAACGACGCCCAACAGTTCTATAGATCSFLTKAQQYYDDFGIAEYNPWFSWAAACGAATTTGAGGACGAATTGGTTTGGGGTGCCTTATGGTTGTACAAGGCTACTATDQKCTSSQLLLAQITQEQQYIDKITAGGATGAGAGTTTCTTAACAAAAGCCCAACAGTACTATGACGATTTTGGAATTGCCGAYCDHMISGQQRTPKGLVYIDTWGSLGTATAATCCTTGGTTCAGTTGGGACCAGAAATGTACTTCCTCACAGTTGTTATTGGCARMAANAAYLCLEAASAGLKPTEYRCAAATTACCCAGGAACAACAATACATAGACAAAATCACTGCTTATTGTGACCATATGAFATEQIGYALGDTGKSFVVGFGVNATTTCAGGACAGCAAAGAACTCCAAAGGGTTTAGTGTACATTGACACTTGGGGTTCTPPSHESHRSSSCPDAPAPCDWVTYGTTGAGAATGGCCGCAAACGCTGCCTACTTATGTTTGGAAGCAGCTTCAGCCGGTTTAASVDPNPHVLYGAIVGGPGPNDEYDDAACCTACAGAGTACAGAGCATTCGCAACAGAACAAATAGGATACGCATTGGGTGATQRYDYVHNEVADDYNAGYQGCLAACAGGAAAATCTTTCGTGGTTGGATTTGGTGTTAACCCACCTTCCCATGAAAGTCACA ALNELGATCATCTTCCTGCCCAGACGCTCCAGCCCCTTGTGATTGGGTAACATATGGTAGTGT(SEQ ID NO: 40)CGATCCAAACCCTCATGTGTTATACGGAGCAATTGTTGGTGGACCAGGTCCTAACGATGAATATGATGACCAGAGATACGATTATGTACACAATGAAGTCGCTGATGACTACAACGCTGGTTATCAAGGATGCCTGGCCGCTTTGAACGAGTTGTAAGGCGCGCC (SEQ ID NO: 20)

When using the methods above, the term “about” is used precisely toaccount for fractional percentages of codon frequencies for a givenamino acid. As used herein, “about” is defined as one amino acid more orone amino acid less than the value given. The whole number value ofamino acids is rounded up if the fractional frequency of usage is 0.50or greater, and is rounded down if the fractional frequency of use is0.49 or less. Using again the example of the frequency of usage ofleucine in human genes for a hypothetical polypeptide having 62 leucineresidues, the fractional frequency of codon usage would be calculated bymultiplying 62 by the frequencies for the various codons. Thus, 7.28percent of 62 equals 4.51 UUA codons, or “about 5,” i.e., 4, 5, or 6 UUAcodons, 12.66 percent of 62 equals 7.85 UUG codons or “about 8,” i.e.,7, 8, or 9 UUG codons, 12.87 percent of 62 equals 7.98 CUU codons, or“about 8,” i.e., 7, 8, or 9 CUU codons, 19.56 percent of 62 equals 12.13CUC codons or “about 12,” i.e., 11, 12, or 13 CUC codons, 7.00 percentof 62 equals 4.34 CUA codons or “about 4,” i.e., 3, 4, or 5 CUA codons,and 40.62 percent of 62 equals 25.19 CUG codons, or “about 25,” i.e.,24, 25, or 26 CUG codons.

Randomly assigning codons at an optimized frequency to encode a givenpolypeptide sequence, can be done manually by calculating codonfrequencies for each amino acid, and then assigning the codons to thepolypeptide sequence randomly. Additionally, various algorithms andcomputer software programs are readily available to those of ordinaryskill in the art. For example, the “EditSeq” function in the LasergenePackage, available from DNAstar, Inc., Madison, Wis., thebacktranslation function in the VectorNTI Suite, available fromInforMax, Inc., Bethesda, Md., and the “backtranslate” function in theGCG—Wisconsin Package, available from Accelrys, Inc., San Diego, Calif.In addition, various resources are publicly available to codon-optimizecoding region sequences, e.g., the “backtranslation” function athttp://www.entelechon.com/bioinformatics/backtranslation.php?lang=eng(visited Apr. 15, 2008) and the “backtranseq” function available athttp://bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html (visited Jul. 9, 2002).Constructing a rudimentary algorithm to assign codons based on a givenfrequency can also easily be accomplished with basic mathematicalfunctions by one of ordinary skill in the art.

A number of options are available for synthesizing codon optimizedcoding regions designed by any of the methods described above, usingstandard and routine molecular biological manipulations well known tothose of ordinary skill in the art. In one approach, a series ofcomplementary oligonucleotide pairs of 80-90 nucleotides each in lengthand spanning the length of the desired sequence are synthesized bystandard methods. These oligonucleotide pairs are synthesized such thatupon annealing, they form double stranded fragments of 80-90 base pairs,containing cohesive ends, e.g., each oligonucleotide in the pair issynthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond theregion that is complementary to the other oligonucleotide in the pair.The single-stranded ends of each pair of oligonucleotides is designed toanneal with the single-stranded end of another pair of oligonucleotides.The oligonucleotide pairs are allowed to anneal, and approximately fiveto six of these double-stranded fragments are then allowed to annealtogether via the cohesive single stranded ends, and then they ligatedtogether and cloned into a standard bacterial cloning vector, forexample, a TOPO® vector available from Invitrogen Corporation, Carlsbad,Calif. The construct is then sequenced by standard methods. Several ofthese constructs consisting of 5 to 6 fragments of 80 to 90 base pairfragments ligated together, i.e., fragments of about 500 base pairs, areprepared, such that the entire desired sequence is represented in aseries of plasmid constructs. The inserts of these plasmids are then cutwith appropriate restriction enzymes and ligated together to form thefinal construct. The final construct is then cloned into a standardbacterial cloning vector, and sequenced. Additional methods would beimmediately apparent to the skilled artisan. In addition, gene synthesisis readily available commercially.

In certain embodiments, an entire polypeptide sequence, or fragment,variant, or derivative thereof is codon optimized by any of the methodsdescribed herein. Various desired fragments, variants or derivatives aredesigned, and each is codon-optimized individually. In addition,partially codon-optimized coding regions of the present invention can bedesigned and constructed. For example, the invention includes a nucleicacid fragment of a codon-optimized coding region encoding a polypeptidein which at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%of the codon positions have been codon-optimized for a given species.That is, they contain a codon that is preferentially used in the genesof a desired species, e.g., a yeast species such as Saccharomycescerevisiae, in place of a codon that is normally used in the nativenucleic acid sequence.

In some embodiments of the present invention, the codon-optimizedpolynucleotide encoding the termite or termite-associated symbiontcellulase is about 85%, about 80%, about 75%, about 70%, about 65%,about 60%, about 55% or about 50% identical to the endogenous codingsequence. In some embodiments the codon-optimized polynucleotideencoding the termite or termite-associated symbiont cellulase is lessthan about 95%, about 90%, about 85%, about 80%, about 75%, about 70%,about 65%, about 60%, about 55%, or about 50% identical to theendogenous coding sequence. In some embodiments, the codon-optimizedpolynucleotide encoding the termite or termite-associated symbiontcellulase is from about 50% to about 95%, from about 60% to about 95%,from about 70% to about 95%, from about 80% to about 95% or from about90% to about 95% identical to the endogenous coding sequence. In someembodiments, the codon-optimized polynucleotide encoding the termite ortermite-associated symbiont cellulase is from about 50% to about 90%,from about 60% to about 90%, from about 70% to about 90% or from about80% to about 90% identical to the endogenous coding sequence. In someembodiments, the codon-optimized polynucleotide encoding the termite ortermite-associated symbiont cellulase is from about 50% to about 85%,from about 60% to about 85% or from about 70% to about 85% identical tothe endogenous coding sequence. In some embodiments, the codon-optimizedpolynucleotide encoding the termite or termite-associated symbiontcellulase is from about 50% to about 80%, from about 60% to about 80% orfrom about 70% to about 80% identical to the endogenous coding sequence.In some embodiments, the codon-optimized polynucleotide encoding thetermite or termite-associated symbiont cellulase is from about 50% toabout 75% or from about 60% to about 75% identical to the endogenouscoding sequence. In some embodiments, the codon-optimized polynucleotideencoding the termite or termite-associated symbiont cellulase is fromabout 50% to about 70% or from about 60% to about 70% identical to theendogenous coding sequence.

In additional embodiments, a full-length polypeptide sequence iscodon-optimized for a given species resulting in a codon-optimizedcoding region encoding the entire polypeptide, and then nucleic acidfragments of the codon-optimized coding region, which encode fragments,variants, and derivatives of the polypeptide are made from the originalcodon-optimized coding region. As would be well understood by those ofordinary skill in the art, if codons have been randomly assigned to thefull-length coding region based on their frequency of use in a givenspecies, nucleic acid fragments encoding fragments, variants, andderivatives would not necessarily be fully codon optimized for the givenspecies. However, such sequences are still much closer to the codonusage of the desired species than the native codon usage. The advantageof this approach is that synthesizing codon-optimized nucleic acidfragments encoding each fragment, variant, and derivative of a givenpolypeptide, although routine, would be time consuming and would resultin significant expense.

The codon-optimized coding regions can be versions encoding a termite ortermite-associated symbiont cellulase or domains, fragments, variants,or derivatives thereof.

Codon optimization is carried out for a particular species by methodsdescribed herein. For example, in certain embodiments codon-optimizedcoding regions encoding termite cellulases or termite-associatedsymbiont cellulases, or domains, fragments, variants, or derivativesthereof that are optimized according to yeast codon usage, e.g.,Saccharomyces cerevisiae, Kluveromyces lactus or both. In particular,the present invention relates to codon-optimized coding regions encodingpolypeptides of termite cellulases or termite-associated symbiontcellulases, or domains, variants or derivatives thereof which have beenoptimized according to yeast codon usage, for example, Saccharomycescerevisiae and Kluveromyces lactus codon usage. Also provided arepolynucleotides, vectors, and other expression constructs comprisingcodon-optimized coding regions encoding termite cellulases ortermite-associated symbiont cellulases, or domains, fragments, variants,or derivatives thereof, and various methods of using suchpolynucleotides, vectors and other expression constructs.

In certain embodiments described herein, a codon-optimized coding regionencoding any of SEQ ID NOs: 21-40, or domain, fragment, variant, orderivative thereof, is optimized according to codon usage in yeast (e.g.Saccharomyces cerevisiae). Alternatively, a codon-optimized codingregion encoding any of SEQ ID NOs: 21-40 may be optimized according tocodon usage in any plant, animal, or microbial species. In certainembodiments, the codon-optimized coding region is a polynucleotidecomprising a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 1-20, or a fragment thereof.

Polynucleotides of the Invention

The present invention provides for polynucleotides comprising a nucleicacid fragment which encodes at least 50 amino acids of a cellulase,wherein said nucleic acid fragment is codon-optimized for expression ina yeast strain and wherein the cellulase is a termite cellulase or atermite-associated cellulase. In some embodiments, the cellulase is acellulase comprising the amino acid sequence of SEQ ID NOs: 21-40, or afragment, variant or derivative thereof. In some embodiments, thecellulase is encoded by a polynucleotide of SEQ ID NOs: 1-20 or afragment, variant or derivative thereof.

The present invention also provides for the use of an isolatedpolynucleotide comprising a nucleic acid at least about 70%, 75%, or 80%identical, at least about 90% to about 95% identical, or at least about96%, 97%, 98%, 99% or 100% identical to any of SEQ ID NOs:1-20, orfragments, variants, or derivatives thereof.

In certain aspects, the present invention relates to a polynucleotidecomprising a nucleic acid encoding a functional or structural domain ofa termite cellulase or termite-associated symbiont cellulase. Thepresent invention also encompasses an isolated polynucleotide comprisinga nucleic acid that is about 70%, 75%, or 80% identical, at least about90% to about 95% identical, or at least about 96%, 97%, 98%, 99% or 100%identical to a nucleic acid encoding a functional or structural domainof a termite cellulase or termite-associated symbiont cellulase.

The present invention also encompasses variants of a termite cellulaseor termite-associated symbiont cellulase. Variants may containalterations in the coding regions, non-coding regions, or both. Examplesare polynucleotide variants containing alterations which produce silentsubstitutions, additions, and/or deletions, but do not alter theproperties or activities of the encoded polypeptide, e.g. the biologicalactivity such as cellulase activity. For example, polynucleotidevariants include one or several nucleic acid deletions, substitutionsand/or additions, where the encoded variant retains cellulase activity.In certain embodiments, nucleotide variants are produced by silentsubstitutions due to the degeneracy of the genetic code. In furtherembodiments, termite cellulase or termite-associated symbiont cellulasepolynucleotide variants can be produced for a variety of reasons, e.g.,to optimize codon expression for a particular host as described above(e.g., change codons in the termite cellulase or termite-associatedsymbiont cellulase mRNA to those preferred by a host such as the yeastSaccharomyces cerevisiae).

The present invention also encompasses an isolated polynucleotidecomprising a nucleic acid that is about 70%, 75%, or 80% identical, atleast about 90% to about 95% identical, or at least about 96%, 97%, 98%,99% or 100% identical to a nucleic acid encoding a fusion protein, wherethe nucleic acid comprises: (1) a first polynucleotide, where the firstpolynucleotide encodes for a termite cellulase or termite-associatedsymbiont cellulase, or domain, fragment, variant, or derivative thereof;and (2) one or more additional polynucleotides, where the one or moreadditional polynucleotides encodes for a termite cellulase ortermite-associated symbiont cellulase, or domain, fragment, variant, orderivative thereof.

In certain embodiments, the nucleic acid encoding a fusion proteincomprises a first polynucleotide encoding for a termite cellulase ortermite-associated symbiont cellulase or domain, fragment, variant orderivative thereof and a second polynucleotide encoding for the S.cerevisiae alpha mating factor signal sequence.

In certain embodiments, the nucleic acid encoding a fusion proteincomprises a first polynucleotide encoding for a termite cellulase ortermite-associated symbiont cellulase and one or more additionalpolynucleotides encoding for a cellulose binding domain (CBM) domain. Inone embodiment, the CBM domain is the CBM domain of T. reesei cbh1 or T.reesei cbh2. The amino acid sequence of the CBM domains of T. reeseiCbh1 and T. reesei Cbh2 are as follows:

T. reesei Cbh1 (SEQ ID NO: 41) HYGQCGGIGYSGPTVCASGTTCQVLNPYYSQCLT. reesei Cbh2 (SEQ ID NO: 42) VYSNDYYSQCLPGAASSSSSTRAASTTSRVSP

In one particular embodiment the nucleic acid encoding a fusion proteinincludes a first polynucleotide that is a codon-optimized termitecellulase or termite-associated symbiont cellulase, and the one or moreadditional polynucleotides encodes for a codon-optimized CBM of T.reesei Cbh1 or Cbh2.

In further embodiments of the fusion protein, the first polynucleotideis either 5′ (i.e. upstream) or 3′ (i.e. downstream) to the one or moreadditional polynucleotides. In certain other embodiments, the firstpolynucleotide and/or the one or more additional polynucleotides areencoded by codon-optimized polynucleotides, for example, polynucleotidescodon-optimized for S. cerevisiae.

Also provided in the present invention are allelic variants, orthologs,and/or species homologs. Procedures known in the art can be used toobtain full-length genes, allelic variants, splice variants, full-lengthcoding portions, orthologs, and/or species homologs of genescorresponding to any of SEQ ID NOs: 1-20, using information from thesequences disclosed herein. For example, allelic variants and/or specieshomologs may be isolated and identified by making suitable probes orprimers from the sequences provided herein and screening a suitablenucleic acid source for allelic variants and/or the desired homologue.

By a nucleic acid having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of the nucleicacid is identical to the reference sequence except that the nucleotidesequence may include up to five point mutations per each 100 nucleotidesof the reference nucleotide sequence encoding the particularpolypeptide. In other words, to obtain a nucleic acid having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The query sequence may bean entire sequence shown of any of SEQ ID NOs:1-20, or any fragment ordomain specified as described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to a nucleotide sequence or polypeptide of the presentinvention can be determined conventionally using known computerprograms. A method for determining the best overall match between aquery sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, can bedetermined using the FASTDB computer program based on the algorithm ofBrutlag et al. (Comp. App. Biosci. (1990) 6:237-245.) In a sequencealignment the query and subject sequences are both DNA sequences. An RNAsequence can be compared by converting U's to T's. The result of saidglobal sequence alignment is in percent identity. Preferred parametersused in a FASTDB alignment of DNA sequences to calculate percentidentity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, JoiningPenalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5,Gap Size Penalty 0.05, Window Size=500 or the length of the subjectnucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to be made forthe purposes of the present invention.

Some embodiments of the invention encompass a nucleic acid moleculecomprising at least 10, 20, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, or 800 consecutive nucleotides or more of anyof SEQ ID NOs:1-20, or domains, fragments, variants, or derivativesthereof.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence encoding SEQ ID NOs: 21-40 or may be a differentcoding sequence which coding sequence, as a result of the redundancy ordegeneracy of the genetic code, encodes the same mature polypeptide asthe DNA of any one of SEQ ID NOs:1-20.

In certain embodiments, the present invention provides an isolatedpolynucleotide comprising a nucleic acid fragment which encodes at least10, at least 20, at least 30, at least 40, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 95, at least 100, at least150, at least 200, at least 250, at least 300 or at least 350 or morecontiguous amino acids of SEQ ID NOs: 21-40.

The polynucleotide encoding for the mature polypeptide comprising theamino acid sequence of SEQ ID NOs:21-40 may include, for example, onlythe coding sequence for the mature polypeptide; the coding sequence ofany domain of the mature polypeptide; the coding sequence for the maturepolypeptide and the coding sequence for a fusion polypeptide; and thecoding sequence for the mature polypeptide (or domain-encoding sequence)together with non-coding sequence, such as introns or non-codingsequence 5′ and/or 3′ of the coding sequence for the mature polypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only sequences encoding for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequences. In some embodiments of the presentinvention, the polynucleotide encodes at least about 100, 150, 200, 250,300 or 350 contiguous amino acids of a termite cellulase or atermite-associated symbiont cellulase.

In further aspects of the invention, nucleic acid molecules havingsequences at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% identical to the nucleic acid sequences disclosed herein, encode apolypeptide having cellulase functional activity. By “a polypeptidehaving cellulase functional activity” is intended polypeptidesexhibiting activity similar, but not necessarily identical, to afunctional activity of the cellulase polypeptides of the presentinvention, as measured, for example, in a particular biological assay.For example, a cellulase functional activity can routinely be measuredby determining the ability of a cellulase polypeptide to hydrolyzecellulose, i.e. by measuring the level of cellulase activity

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large portion of thenucleic acid molecules having a sequence at least about 90%, 95%, 96%,97%, 98%, or 99% identical to the nucleic acid sequence of any of SEQ IDNOs:1-20, or fragments thereof, will encode polypeptides “havingcellulase functional activity.” In fact, since degenerate variants ofany of these nucleotide sequences all encode the same polypeptide, inmany instances, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving cellulase functional activity.

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNAs which have a high sequence similarity tothe termite cellulase and termite-associated symbiont cellulase genes ofthe present invention, or a gene encoding for a protein with similarbiological activity. The probe length can vary from 5 bases to tens ofthousands of bases, and will depend upon the specific test to be done.Typically a probe length of about 15 bases to about 30 bases issuitable. Only part of the probe molecule need be complementary to thenucleic acid sequence to be detected. In addition, the complementaritybetween the probe and the target sequence need not be perfect.Hybridization does occur between imperfectly complementary moleculeswith the result that a certain fraction of the bases in the hybridizedregion are not paired with the proper complementary base.

In certain embodiments, a hybridization probe may have at least 30 basesand may contain, for example, 50 or more bases. The probe may also beused to identify a cDNA clone corresponding to a full length transcriptand a genomic clone or clones that contain the complete gene includingregulatory and promoter regions, exons, and introns. An example of ascreen comprises isolating the coding region of the gene by using theknown DNA sequence to synthesize an oligonucleotide probe. Labeledoligonucleotides having a sequence complementary to that of the gene ofthe present invention are used to screen a library of bacterial orfungal cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least about 70%,at least about 90%, or at least about 95% identity between thesequences. The present invention particularly relates to polynucleotideswhich hybridize under stringent conditions to the hereinabove-describedpolynucleotides. As herein used, the term “stringent conditions” meanshybridization will occur only if there is at least about 95% or at leastabout 97% identity between the sequences. In certain aspects of theinvention, the polynucleotides which hybridize to the hereinabovedescribed polynucleotides encode polypeptides which either retainsubstantially the same biological function or activity as the maturepolypeptide encoded by the DNAs of any of SEQ ID NOs:1-20.

Alternatively, polynucleotides which hybridize to thehereinabove-described sequences may have at least 20 bases, at least 30bases, or at least 50 bases which hybridize to a polynucleotide of thepresent invention and which has an identity thereto, as hereinabovedescribed, and which may or may not retain activity. For example, suchpolynucleotides may be employed as probes for the polynucleotide of anyof SEQ ID NOs: 1-20, for example, for recovery of the polynucleotide oras a diagnostic probe or as a PCR primer.

Hybridization methods are well defined and have been described above.Nucleic acid hybridization is adaptable to a variety of assay formats.One of the most suitable is the sandwich assay format. The sandwichassay is particularly adaptable to hybridization under non-denaturingconditions. A primary component of a sandwich-type assay is a solidsupport. The solid support has adsorbed to it or covalently coupled toit immobilized nucleic acid probe that is unlabeled and complementary toone portion of the sequence.

For example, genes encoding similar proteins or polypeptides to those ofthe instant invention could be isolated directly by using all or aportion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired bacteria using methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (see, e.g., Maniatis, 1989).Moreover, the entire sequences can be used directly to synthesize DNAprobes by methods known to the skilled artisan such as random primersDNA labeling, nick translation, or end-labeling techniques, or RNAprobes using available in vitro transcription systems.

In certain aspects of the invention, polynucleotides which hybridize tothe hereinabove-described sequences having at least 20 bases, at least30 bases, or at least 50 bases which hybridize to a polynucleotide ofthe present invention may be employed as PCR primers. Typically, inPCR-type amplification techniques, the primers have different sequencesand are not complementary to each other. Depending on the desired testconditions, the sequences of the primers should be designed to providefor both efficient and faithful replication of the target nucleic acid.Methods of PCR primer design are common and well known in the art.Generally two short segments of the instant sequences may be used inpolymerase chain reaction (PCR) protocols to amplify longer nucleic acidfragments encoding homologous genes from DNA or RNA. The polymerasechain reaction may also be performed on a library of cloned nucleic acidfragments wherein the sequence of one primer is derived from the instantnucleic acid fragments, and the sequence of the other primer takesadvantage of the presence of the polyadenylic acid tracts to the 3′ endof the mRNA precursor encoding microbial genes. Alternatively, thesecond primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al., PNAS USA 85:8998 (1988)) to generate cDNAs byusing PCR to amplify copies of the region between a single point in thetranscript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′directions can be designed from the instant sequences. Usingcommercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or5′ cDNA fragments can be isolated (Ohara et al., PNAS USA 86:5673(1989); Loh et al., Science 243:217 (1989)).

In addition, specific primers can be designed and used to amplify a partof or full-length of the instant sequences. The resulting amplificationproducts can be labeled directly during amplification reactions orlabeled after amplification reactions, and used as probes to isolatefull length DNA fragments under conditions of appropriate stringency.

Therefore, the nucleic acid sequences and fragments thereof of thepresent invention may be used to isolate genes encoding homologousproteins from the same or other fungal species or bacterial species.Isolation of homologous genes using sequence-dependent protocols is wellknown in the art. Examples of sequence-dependent protocols include, butare not limited to, methods of nucleic acid hybridization, and methodsof DNA and RNA amplification as exemplified by various uses of nucleicacid amplification technologies (e.g., polymerase chain reaction, Mulliset al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR) (Tabor, S.et al., Proc. Acad. Sci. USA 82, 1074, (1985)); or strand displacementamplification (SDA, Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89,392, (1992)).

The polynucleotides of the present invention can also comprise nucleicacids encoding a termite cellulase or termite-associated symbiontcellulase, or domain, fragment, variant, or derivative thereof, fused inframe to a marker sequence which allows for detection of the polypeptideof the present invention. The marker sequence may be a yeast selectablemarker selected from the group consisting of URA3, HIS3, LEU2, TRP1,LYS2, ADE2 or SMR1. Additional marker sequences include otherauxotrophic markers or dominant markers known to one of ordinary skillin the art such as ZEO (zeocin), NEO (G418), hyromycin, arsenite, HPH,NAT and the like.

Polypeptides of the Invention

The present invention further relates to the expression of termitecellulase or termite-associated symbiont cellulase polypeptides in ayeast host cell, such as Saccharomyces cerevisiae. The sequences ofseveral examples of termite cellulase or termite-associated symbiontcellulase polypeptides are set forth above and summarized in Table 3.

The present invention further encompasses polypeptides which comprise,or alternatively consist of, an amino acid sequence which is at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, for example,the polypeptide sequence shown in SEQ ID NOs: 21-40, and/or domains,fragments, variants, or derivative thereof, of any of these polypeptides(e.g., those fragments described herein, or domains of any of SEQ IDNOs: 21-40).

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, (indels) or substituted withanother amino acid. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequence of SEQ ID NOs: 21-40 can be determinedconventionally using known computer programs. As discussed above, amethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). In a sequence alignment the query andsubject sequences are either both nucleotide sequences or both aminoacid sequences. The result of said global sequence alignment is inpercent identity. Preferred parameters used in a FASTDB amino acidalignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, JoiningPenalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter. Also as discussed above, manual corrections may be made to theresults in certain instances.

In certain embodiments, the polypeptide of the present inventionencompasses a fusion protein comprising a first polypeptide, where thefirst polypeptide is a termite cellulase or a termite-associatedsymbiont cellulase or domain, fragment, variant, or derivative thereof,and one or more additional polypeptides. In some embodiments the one ormore additional polypeptides is a signal sequence. The signal sequencecan be from any organism. For example, in some embodiments, the one ormore additional polypeptides is an S. cerevisiae polypeptide. In oneparticular embodiment, the S. cerevisiae polypeptide is the S.cerevisiae alpha mating factor signal sequence. In some embodiments thesignal sequence comprises the amino acid sequence MRFPSIFTAVLFAASSALA(SEQ ID NO: 43).

In certain embodiments, the polypeptide of the present inventionencompasses a fusion protein comprising a first polypeptide, where thefirst polypeptide is a termite cellulase or a termite-associatedsymbiont cellulase or domain, fragment, variant, or derivative thereof,and one or more additional polypeptides, where the one or moreadditional polypeptides comprises a cellulose binding domain (CBM). Insome embodiments, the CBM is Neosartorya fischeri Cbh1, H. grisea Cbh1,Chaetomium thermophilum Cbh1, T. reesei Cbh1 or T. reesei Cbh2, or adomain, fragment, variant, or derivative thereof.

In further embodiments of the fusion protein, the first polypeptide iseither N-terminal or C-terminal to the one or more additionalpolypeptides. In certain other embodiments, the first polypeptide and/orthe one or more additional polypeptides are encoded by codon-optimizedpolynucleotides, for example, polynucleotides codon-optimized forexpression in S. cerevisiae. In particular embodiments, the firstpolynucleotide is a codon-optimized termite cellulase or atermite-associated symbiont cellulase and the one or more additionalpolynucleotides encodes for a codon-optimized CBM from T. reesei Cbh1 orCbh2. In certain other embodiments, the first polypeptide and the one ormore additional polypeptides are fused via a linker sequence.

In certain aspects of the invention, the polypeptides andpolynucleotides of the present invention are provided in an isolatedform, e.g., purified to homogeneity.

The present invention also encompasses polypeptides which comprise, oralternatively consist of, an amino acid sequence which is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% similar to a polypeptide comprisingthe amino acid sequence of any of SEQ ID NOs: 21-40, and to portions ofsuch polypeptide with such portion of the polypeptide generallycontaining at least 30 amino acids and more preferably at least 50 aminoacids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and conserved amino acidsubstitutes thereto of the polypeptide to the sequence of a secondpolypeptide.

The present invention further relates to a domain, fragment, variant,derivative, or analog of the polypeptide comprising the amino acidsequence of any of SEQ ID NOs: 21-40.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis, therefore, the fragments may be employed asintermediates for producing the full-length polypeptides.

Fragments of termite cellulase and termite-associated symbiont cellulasepolypeptides of the present invention encompass domains, proteolyticfragments, deletion fragments and in particular, fragments of termitecellulase and termite-associated symbiont cellulase polypeptides whichretain any specific biological activity of the cellulase protein.Polypeptide fragments further include any portion of the polypeptidewhich comprises a catalytic activity of the cellulase protein.

The variant, derivative or analog of the polypeptide comprising theamino acid sequence of any of SEQ ID NOs: 21-40, can be (i) one in whichone or more of the amino acid residues are substituted with a conservedor non-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide for purification of the polypeptideor (v) one in which a fragment of the polypeptide is soluble, i.e., notmembrane bound, yet still binds ligands to the membrane bound receptor.Such variants, derivatives and analogs are deemed to be within the scopeof those skilled in the art from the teachings herein.

The polypeptides of the present invention further include variants ofthe polypeptides. A “variant” of the polypeptide can be a conservativevariant, or an allelic variant. As used herein, a conservative variantrefers to alterations in the amino acid sequence that does not adverselyaffect the biological functions of the protein. A substitution,insertion or deletion is said to adversely affect the protein when thealtered sequence prevents or disrupts a biological function associatedwith the protein. For example, the overall charge, structure orhydrophobic-hydrophilic properties of the protein can be altered withoutadversely affecting a biological activity. Accordingly, the amino acidsequence can be altered, for example to render the peptide morehydrophobic or hydrophilic, without adversely affecting the biologicalactivities of the protein.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a side chain witha similar charge. Families of amino acid residues having side chainswith similar charges have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity (e.g.,cellulase activity).

By an “allelic variant” is intended alternate forms of a gene occupyinga given locus on a chromosome of an organism. Genes II, Lewin, B., ed.,John Wiley & Sons, New York (1985). Non-naturally occurring variants maybe produced using art-known mutagenesis techniques. Allelic variants,though possessing a slightly different amino acid sequence than thoserecited above, will still have the same or similar biological functionsassociated with the termite cellulase or termite-associated symbiontcellulase protein.

In some embodiments, the allelic variants, the conservative substitutionvariants, and members of the termite cellulase or termite-associatedsymbiont cellulase protein family, will have an amino acid sequencehaving at least 75%, at least 80%, at least 90%, at least 95% amino acidsequence identity with a termite cellulase or termite-associatedsymbiont cellulase amino acid sequence set forth in any one of SEQ IDNOs:21-40. Identity or homology with respect to such sequences isdefined herein as the percentage of amino acid residues in the candidatesequence that are identical with the known peptides, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent homology, and not considering any conservative substitutions aspart of the sequence identity. N terminal, C terminal or internalextensions, deletions, or insertions into the peptide sequence shall notbe construed as affecting homology.

Thus, the proteins and peptides of the present invention includemolecules comprising the amino acid sequence of SEQ ID NOs: 21-40 orfragments thereof having a consecutive sequence of at least about 3, 4,5, 6, 10, 15, 20, 25, 30, 35 or more amino acid residues of the termitecellulase or termite-associated symbiont cellulase polypeptide sequence;amino acid sequence variants of such sequences wherein at least oneamino acid residue has been inserted N- or C-terminal to, or within, thedisclosed sequence; amino acid sequence variants of the disclosedsequences, or their fragments as defined above, that have beensubstituted by another residue. Contemplated variants further includethose containing predetermined mutations by, e.g., homologousrecombination, site-directed or PCR mutagenesis, and the correspondingproteins of other animal species, the alleles or other naturallyoccurring variants of the family of proteins, and derivatives whereinthe protein has been covalently modified by substitution, chemical,enzymatic, or other appropriate means with a moiety other than anaturally occurring amino acid (for example, a detectable moiety such asan enzyme or radioisotope).

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the termite or termite-associated symbiont cellulase.For instance, one or more amino acids can be deleted from the N-terminusor C-terminus of the secreted protein without substantial loss ofbiological function.

Thus, the invention further includes termite cellulase ortermite-associated symbiont cellulase polypeptide variants which showsubstantial biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity.Polypeptide variants of the invention further include one or severalamino acid deletions, substitutions and/or additions, where the variantretains substantial biological activity. For example, polypeptidevariants include one or several amino acid deletions, substitutionsand/or additions, where the variant retains cellulase activity.

The skilled artisan is fully aware of amino acid substitutions that areeither less likely or not likely to significantly effect proteinfunction (e.g., replacing one aliphatic amino acid with a secondaliphatic amino acid), as further described below.

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie et al., “Deciphering the Messagein Protein Sequences: Tolerance to Amino Acid Substitutions,” Science247:1306-1310 (1990), wherein the authors indicate that there are twomain strategies for studying the tolerance of an amino acid sequence tochange.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, conserved amino acids can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions where substitutions havebeen tolerated by natural selection indicates that these positions arenot critical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

As the authors state, these two strategies have revealed that proteinsare often surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved. Moreover, tolerated conservative aminoacid substitutions involve replacement of the aliphatic or hydrophobicamino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residuesSer and Thr; replacement of the acidic residues Asp and Glu; replacementof the amide residues Asn and Gln, replacement of the basic residuesLys, Arg, and His; replacement of the aromatic residues Phe, Tyr, andTrp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met,and Gly.

The terms “derivative” and “analog” refer to a polypeptide differingfrom the termite cellulase or termite-associated symbiont cellulasepolypeptide, but retaining essential properties thereof. Generally,derivatives and analogs are overall closely similar, and, in manyregions, identical to the termite cellulase or termite-associatedsymbiont cellulase polypeptides. The term “derivative” and “analog” whenreferring to termite cellulase or termite-associated symbiont cellulasepolypeptides of the present invention include any polypeptides whichretain at least some of the activity of the corresponding nativepolypeptide, e.g., the endogluconase activity, exogluconase activity,β-glucosidase activity or the activity of the catalytic domain of one ofthese proteins.

Derivatives of termite cellulase or termite-associated symbiontcellulase polypeptides of the present invention, are polypeptides whichhave been altered so as to exhibit additional features not found on thenative polypeptide. Derivatives can be covalently modified bysubstitution, chemical, enzymatic, or other appropriate means with amoiety other than a naturally occurring amino acid (for example, adetectable moiety such as an enzyme or radioisotope). Examples ofderivatives include fusion proteins.

An analog is another form of a termite cellulase or termite-associatedsymbiont cellulase polypeptide of the present invention. An “analog”also retains substantially the same biological function or activity asthe polypeptide of interest, i.e., functions as a cellobiohydrolase. Ananalog includes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

Vectors Encoding Termite Cellulases and/or Termite-Associated SymbiontCellulases

The present invention also relates to vectors which includepolynucleotides of the present invention. Vectors of the presentinvention may be, for example, a cloning vector for example, in the formof a plasmid, a viral particle, a phage, etc. In addition, thepolynucleotides of the present invention may be employed for producingpolypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; and yeast plasmids. Both episomal vectors (such aspMU451) and integrative vectors (such as pMU562) can be used. The vectorpMU562 is an integrative yeast expression vector that includes thefollowing sequences: the intergenic region of phage f1; the pMB1replicon responsible for the replication of phagemid; the gene codingfor beta-lactamase that confers resistance to ampicillin; S. cerevisiaedelta integration sites; S. cerevisiae ENO1 promoter; S. cerevisiae ENO1terminator; S. cerevisiae TEF1 promoter; S. cerevisiae TEF1 terminator;Streptoalloteichus hindustanus ble Zeocin resistance gene; and Crerecombinase recognition site. Furthermore, any other vector that can bemaintained in a host cell and allow for gene expression can be used.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively associated withan appropriate expression control sequence(s) (promoter) to direct mRNAsynthesis.

Representative examples of such promoters are as follows:

TABLE 4 Exemplary Promoters Gene Organism Systematic name Reason foruse/benefits PGK1 S. cerevisiae YCR012W Strong constitutive promoterENO1 S. cerevisiae YGR254W Strong constitutive promoter TDH3 S.cerevisiae YGR192C Strong constitutive promoter TDH2 S. cerevisiaeYJR009C Strong constitutive promoter TDH1 S. cerevisiae YJL052W Strongconstitutive promoter ENO2 S. cerevisiae YHR174W Strong constitutivepromoter GPM1 S. cerevisiae YKL152C Strong constitutive promoter TPI1 S.cerevisiae YDR050C Strong constitutive promoter

Additionally, promoter sequences from stress and starvation responsegenes are useful in the present invention. In some embodiments, promoterregions from the S. cerevisiae genes GAC1, GET3, GLC7, GSH1, GSH2, HSF1,HSP12, LCB5, LRE1, LSP1, NBP2, PIL1, PIM1, SGT2, SLG1, WHI2, WSC2, WSC3,WSC4, YAP1, YDC1, HSP104, HSP26, ENA1, MSN2, MSN4, SIP2, SIP4, SIP5,DPL1, IRS4, KOG1, PEP4, HAP4, PRB1, TAX4, ZPR1, ATG1, ATG2, ATG10,ATG11, ATG12, ATG13, ATG14, ATG15, ATG16, ATG17, ATG18, and ATG19 can beused. Any suitable promoter to drive gene expression in the host cellsof the invention can be used.

Additionally the E. coli, lac or trp, and other promoters are known tocontrol expression of genes in prokaryotic or lower eukaryotic cells.Promoter regions can be selected from any desired gene. Particular namedyeast promoters include the constitute promoter ENO1, the PGK1 promoter,the TEF1 promoter and the HXT7 promoter. Particular named bacterialpromoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp.Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.

The expression vector also contains a ribosome binding site fortranslation initiation and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression, or mayinclude additional regulatory regions.

In addition, the expression vectors may contain one or more sequencesencoding selectable marker to provide a phenotypic trait for selectionof transformed host cells such as URA3, HIS3, LEU2, TRP1, LYS2, ADE2,dihydrofolate reductase or neomycin (G418) resistance for eukaryoticcell culture, or tetracycline or ampicillin resistance in E. coli.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In one aspect of this embodiment, theconstruct further comprises regulatory sequences, including, forexample, a promoter, operably associated to the sequence. Large numbersof suitable vectors and promoters are known to those of skill in theart, and are commercially available. The following vectors are providedby way of example.

The vector containing the appropriate DNA sequence as herein, as well asan appropriate promoter or control sequence, can be employed totransform an appropriate host to permit the host to express the protein.

Thus, in certain aspects, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, e.g., Saccharomyces cerevisiae, or the host cellcan be a prokaryotic cell, such as a bacterial cell.

Representative examples of appropriate hosts include bacterial cells,such as E. coli, Streptomyces, Salmonella typhimurium; thermophilic ormesophlic bacteria; fungal cells, such as yeast; and plant cells, etc.The selection of an appropriate host is deemed to be within the scope ofthose skilled in the art from the teachings herein.

Appropriate fungal hosts include yeast. In certain aspects of theinvention the yeast is Saccharomyces cerevisiae, Kluveromyces lactus,Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichiastipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma,Candida utilis, Arxula adeninivorans, Debaryomyces hansenii,Debaryomyces polymorphus, Schwanniomyces occidentalis, Issatchenkiaorientalis, or Kluveromyces marxianus.

Yeast: Yeast vectors include those of five general classes, based ontheir mode of replication in yeast, YIp (yeast integrating plasmids),YRp (yeast replicating plasmids), YCp (yeast replicating plasmids withcentromere (CEN) elements incorporated), YEp (yeast episomal plasmids),and YLp (yeast linear plasmids). With the exception of the YLp plasmids,all of these plasmids can be maintained in E. coli as well as inSaccharomyces cerevisiae and thus are also referred to as yeast shuttlevectors.

In certain aspects, these plasmids can contain types of selectable genesincluding plasmid-encoded drug-resistance genes and/or cloned yeastgenes, where the drug resistant gene and/or cloned yeast gene can beused for selection. Drug-resistance genes include, e.g., ampicillin,kanamycin, tetracycline, neomycin, hygromycin, zeocin, NAT, arsentiedand sulfometuron methyl. Cloned yeast genes include e.g., HIS3, LEU2,LYS2, TRP1, URA3, TRP1 and SMR1. pYAC vectors may also be utilized toclone large fragments of exogenous DNA on to artificial linearchromosomes

In certain aspects of the invention, YCp plasmids, which have highfrequencies of transformation and increased stability to due theincorporated centromere elements, are utilized. In certain other aspectsof the invention, YEp plasmids, which provide for high levels of geneexpression in yeast, are utilized. In additional aspects of theinvention, YRp plasmids are utilized.

The vector can also contain one or more polynucleotides. The one or morepolynucleotides can, for example, encode one or more cellulases. The oneor more cellulases can be one or more endogluconases, such asendogluconase I, an exogluconase, such as cellobiohyrolase I orcellobiohydrolase II or a β-glucosidase, such as β-glucosidase I. Theone or more polynucleotides can be a termite or termite-associatedsymbiont polynucleotide, or can be a polynucleotide from anotherorganism, for example from T. reesei, S. fibuligera, Neosartoryafisheri, Chaetomium thermophilum or T. emersonni. In some embodiments,the one or more polynucleotides encodes a termite or termite-associatedsymbiont cellulase or a cellulase from another organism such as T.reesei, S. fibuligera, Neosartorya fisheri, Chaetomium thermophilum orT. emersonni.

In one embodiment of the present invention, one of the one or morepolynucleotides encodes the Schizochytrium aggregatum Cbh1 polypeptide,or a fragment, variant or derivative thereof. The amino acid sequence ofthe Schizochytrium aggregatum Cbh1 polypeptide corresponds to SEQ ID NO:44 as follows:

(SEQ ID NO: 44) MSAITLALGALALSSVVNAQQAGTLTPEKHPAFSVSTCSAGGTCTSKTQSIVLDGNWRWLHSTSGSTNCYTGNTFDKTLCPDGVTCAANCALDGADYTGTYGIKASGNSLSLQLKTGSNVGSRVYLMDEQDKNYQLFNLKNQEFTFDVDVSKIGCGLNGALYFVSMPADGGLSTTNKAGTKFGTGYCDAQCPKDIKFIKGKANSDGWTASSNNANTGFGTTGSCCNEMDIWEANGISNAVTPHSCSPGNAACTSDTTCGSGDGNRYKGYCDKDGCDFNPFRMGNQTFYGPGKTIDTTKPLTVVTQFITSDNTASGDLVEIRRKYVQGGKVFDQPTSNVAGVSGNSITDTFCKNQKSVFGDTNDFAAKGGLKAMGDAFADGMVLVMSLWDDYDVNMHWLNSPYPTDADPTKPGVARGTCSITSGKPADVESQTPGATVVYSNIKTGPIGSTFSGAQQPGGPGSGSSSSSSAGGSSTTSRSSSTTSRATTTSVGTTTTTTSSRTTTTSAAGGVVQKYGQCGGLTYTGPTTCVSGTTCTKANDYYSQCL

In one particular embodiment, one of the one or more polynucleotidescomprises the cDNA sequence encoding Schizochytrium aggregatum cbh1, ora fragment, derivative or variant thereof. The cDNA sequence encodingthe Schizochytrium aggregatum cbh1 is as follows:

(SEQ ID NO: 45) ATGTCTGCCATTACCCTCGCCCTGGGTGCTCTTGCCCTCAGCTCTGTTGTCAACGCTCAGCAGGCTGGAACCCTTACTCCTGAAAAACACCCTGCTTTTTCTGTGTCTACTTGCTCTGCCGGCGGCACTTGCACGTCCAAGACCCAGAGCATTGTGCTCGATGGCAACTGGCGCTGGCTCCACTCTACTTCCGGCTCCACCAACTGCTACACAGGTAACACCTTCGACAAGACTTTGTGCCCTGATGGAGTGACTTGCGCCGCAAACTGCGCCCTCGATGGTGCTGACTACACCGGCACTTACGGTATCAAGGCATCCGGCAACTCTCTGAGCCTTCAGCTCAAGACTGGCAGCAACGTTGGCTCCAGAGTCTACCTCATGGACGAGCAGGACAAGAACTACCAGCTCTTCAACCTGAAGAACCAGGAGTTTACGTTCGACGTCGACGTCAGCAAGATCGGATGTGGTCTCAACGGCGCTCTGTACTTCGTGTCCATGCCCGCAGATGGTGGACTTTCTACCACTAACAAGGCCGGCACCAAGTTCGGAACAGGATATTGTGATGCTCAGTGTCCTAAAGACATCAAGTTTATCAAGGGCAAGGCAAACAGCGATGGCTGGACAGCATCTTCCAACAACGCAAACACCGGTTTCGGTACGACCGGCTCCTGCTGCAACGAGATGGATATCTGGGAGGCAAACGGGATCTCCAACGCTGTGACTCCTCACTCCTGCAGTCCCGGCAACGCCGCTTGCACTTCTGACACAACTTGTGGCTCTGGCGACGGTAACCGCTACAAAGGCTACTGTGACAAGGACGGTTGCGATTTCAACCCCTTCAGGATGGGCAACCAGACCTTCTACGGCCCCGGCAAGACTATCGACACCACCAAGCCTCTCACTGTGGTCACCCAATTCATTACCTCTGACAACACTGCTAGTGGCGATCTTGTTGAGATCCGTCGCAAGTACGTCCAGGGCGGCAAGGTCTTCGATCAGCCCACATCCAACGTTGCTGGCGTTAGCGGCAACTCGATCACCGACACCTTCTGCAAAAACCAGAAGTCCGTCTTCGGTGACACTAACGACTTCGCTGCGAAGGGTGGCTTGAAGGCTATGGGCGACGCCTTCGCTGATGGCATGGTCCTTGTCATGTCTCTGTGGGATGATTACGATGTCAACATGCACTGGCTCAACTCTCCTTACCCAACTGACGCCGACCCAACAAAGCCTGGTGTTGCCCGTGGAACTTGCTCTATCACCTCTGGTAAGCCCGCCGACGTCGAGAGCCAGACTCCTGGTGCCACCGTTGTCTACTCGAACATCAAGACTGGTCCCATTGGCTCCACCTTCTCTGGCGCCCAACAGCCCGGTGGCCCCGGCAGTGGTTCTTCATCTTCCAGCTCAGCGGGAGGCTCAAGCACCACCTCCAGGTCTTCTTCTACCACCTCCAGGGCTACCACCACGAGTGTCGGGACCACTACCACCACCACTAGCTCTCGCACGACCACAACCAGCGCTGCTGGCGGCGTCGTCCAGAAGTACGGACAGTGCGGTGGCCTGACATACACTGGTCCTACTACTTGTGTGAGCGGAACCACTTGCACCAAGGCCAACGACTACTACTCGCAGTGCTTG.

In another particular embodiment, the one or more polynucleotidescomprises a codon-optimized version of the cDNA sequence encodingSchizochytrium aggregatum cbh1, or a fragment, derivative or variantthereof. For example, a codon-optimized cDNA sequence encoding theSchizochytrium aggregatum cbh1 can comprise the sequence of SEQ ID NO:46as follows, or a fragment, variant or derivative thereof. In SEQ IDNO:46, the Schizochytrium aggregatum cbh1 cDNA sequence has been codonoptimized for expression in Saccharomyces cerevisiae. The nativeSchizochytrium aggregatum cbh1 signal sequence is exchanged by replacingit with a slightly modified (one amino acid different) Saccharomycescerevisiae alpha mating factor pre signal sequence (underlined). TheSTOP-codon is double underlined in the sequence shown below.

(SEQ ID NO: 46) ATGAGATTTCCATCTATTTTCACTGCTGTTTTGTTCGCAGCCTCATCGAGTCTAGCTCAACAGGCCGGTACTCTAACGCCTGAGAAACATCCCGCCTTCTCCGTTAGTACATGTTCCGCTGGAGGCACGTGCACTAGTAAGACACAAAGCATAGTCTTAGATGGCAACTGGAGATGGCTTCACAGCACATCCGGTTCAACGAACTGTTATACTGGCAATACATTCGACAAGACGCTTTGTCCCGATGGTGTCACTTGTGCCGCTAATTGTGCTTTGGACGGTGCAGACTATACCGGAACGTATGGCATAAAGGCTTCAGGAAATTCCTTATCCCTACAGCTTAAAACTGGAAGTAATGTGGGTTCTAGAGTTTACTTGATGGACGAGCAAGATAAGAATTATCAATTATTCAACTTGAAGAATCAGGAGTTCACTTTTGATGTAGACGTGTCAAAGATCGGCTGTGGTTTAAACGGCGCCTTGTACTTCGTGTCCATGCCAGCAGACGGAGGTTTGTCCACAACTAACAAAGCTGGTACGAAGTTCGGCACGGGATATTGTGACGCCCAATGCCCAAAAGATATTAAGTTCATCAAAGGAAAGGCAAATTCTGATGGCTGGACAGCTTCCTCAAATAATGCCAACACAGGATTCGGCACAACCGGTAGTTGTTGCAATGAAATGGATATATGGGAAGCAAACGGAATTAGTAATGCTGTTACACCTCATTCATGTTCTCCTGGAAATGCCGCATGTACGTCCGATACGACTTGCGGTAGTGGTGACGGAAACAGATACAAAGGCTATTGCGATAAGGATGGATGCGACTTTAATCCATTCAGAATGGGAAATCAAACTTTCTACGGCCCCGGAAAGACGATAGATACTACGAAGCCACTAACGGTGGTGACACAGTTCATAACGTCAGACAATACAGCTTCTGGCGACTTAGTTGAAATTAGAAGAAAGTATGTGCAAGGAGGTAAAGTGTTTGATCAGCCCACCAGCAACGTAGCCGGTGTCAGTGGCAATTCAATTACAGACACTTTTTGCAAGAACCAGAAATCTGTGTTTGGAGATACGAATGACTTCGCAGCTAAGGGCGGATTAAAAGCAATGGGAGATGCATTTGCTGATGGTATGGTCCTAGTAATGTCCTTATGGGACGATTACGACGTCAATATGCATTGGCTTAATTCACCTTATCCAACCGATGCCGACCCTACAAAGCCAGGTGTTGCTAGAGGTACATGCAGTATCACTAGTGGAAAGCCCGCTGATGTGGAGAGCCAAACCCCTGGTGCTACAGTTGTATACTCAAACATTAAGACTGGTCCAATTGGCTCTACGTTCAGTGGAGCCCAGCAACCTGGAGGCCCCGGATCTGGTTCCTCAAGTAGTTCATCCGCAGGCGGTTCATCCACTACGTCAAGGTCCAGTAGCACTACCTCTAGAGCTACAACTACCAGCGTCGGAACAACCACTACGACAACCTCTAGTAGGACGACCACTACAAGCGCCGCAGGCGGTGTAGTTCAGAAATATGGCCAGTGTGGAGGTCTAACTTACACAGGACCAACGACTTGCGTATCTGGTACAACGTGCACGAAGGCTAATGATTATTACTCCCAATGTTTATAA

In certain embodiments, the vector comprises a (1) a firstpolynucleotide, where the first polynucleotide encodes for a termitecellulase or termite-associated symbiont cellulase, or domain, fragment,variant, or derivative thereof; and (2) one or more additionalpolynucleotides, where the one or more additional polynucleotidesencodes for a termite cellulase or termite-associated symbiontcellulase, or domain, fragment, variant, or derivative thereof.

In certain additional embodiments, the vector comprises a firstpolynucleotide encoding for a termite cellulase or termite-associatedsymbiont cellulase and one or more additional polynucleotides encodingfor the S. cerevisiae alpha mating factor signal sequence or any othersignal sequence.

In certain additional embodiments, the vector comprises a firstpolynucleotide encoding for a termite cellulase or termite-associatedsymbiont cellulase and one or more additional polynucleotides encodingfor the CBM domain. In some embodiments, the CBM domain is the CBMdomain of T. reesei cbh1 or T. reesei cbh2.

In further embodiments, the first and one or more additionalpolynucleotides are in the same orientation, or the one or moreadditional polynucleotides is in the reverse orientation of the firstpolynucleotide. In additional embodiments, the first polynucleotide iseither 5′ (i.e. upstream) or 3′ (i.e. downstream) to the one or moreadditional polynucleotides. In certain other embodiments, the firstpolynucleotide and/or the one or more additional polynucleotides areencoded by codon-optimized polynucleotides, for example, polynucleotidescodon-optimized for S. cerevisiae. In additional embodiments, the firstpolynucleotide is a codon-optimized termite cellulase ortermite-associated symbiont cellulase and the one or more additionalpolynucleotides encodes for a codon-optimized CBM from T. reesei Cbh1 orCbh2.

In particular embodiments, the vector of the present invention is apMU451 plasmid containing a termite cellulase or termite-associatedsymbiont cellulase encoding sequence. A diagram of pMU451 is found inFIG. 1.

However, any other plasmid or vector may be used as long as they are canbe maintained in a host cell and are useful for gene expression.Selection of the appropriate vector and promoter is well within thelevel of ordinary skill in the art.

Heterologous Expression of Termite Cellulases and Termite-AssociatedSymbiont Cellulases in Host Cells and Uses Thereof

In order to address the limitations of the previous systems, the presentinvention provides termite cellulase or termite-associated symbiontcellulase polynucleotides and polypeptides, or domains, variants, orderivatives thereof, that can be effectively and efficiently utilized ina consolidated bioprocessing system. One aspect of the invention, isthus related to the efficient production of cellulases, especiallytermite and termite-associated symbiont cellulases in a host organism.The present invention therefore relates to host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector comprising a sequenceencoding a termite cellulase and/or a termite-associate symbiontcellulase. In certain aspects, the present invention relates to hostcells containing the above-described polynucleotide constructs. In someembodiments, the host cell comprises a polynucleotide that encodes atermite or termite-associated symbiont cellulase or a fragment, variantor derivative thereof. In some embodiments, the polynucleotide iscodon-optimized for expression in a heterologous system. The host cellcan be a higher eukaryotic cell, such as a mammalian cell, or a lowereukaryotic cell, such as a yeast cell, e.g., Saccharomyces cerevisiae.The selection of an appropriate host is deemed to be within the scope ofthose skilled in the art from the teachings herein.

Appropriate hosts include yeast. In certain aspects of the invention theyeast is Saccharomyces cerevisiae, Kluveromyces lactus, Kluveromycesmarxianus, Schizzosaccharomyces pombe, Candida albicans, Pichiapastoris, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorpha,Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyceshansenii, Debaryomyces polymorphus or Schwanniomyces occidentalis.

Introduction of the construct into a host yeast cell, e.g.,Saccharomyces cerevisiae, can be effected by lithium acetatetransformation, spheroplast transformation, or transformation byelectroporation, as described in Current Protocols in Molecular Biology,13.7.1-13.7.10.

Introduction of the construct in other host cells can be effected bycalcium phosphate transfection, DEAF-Dextran mediated transfection, orelectroporation. (Davis, L., et al., Basic Methods in Molecular Biology,(1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Following creation of a suitable host cell and growth of the host cellto an appropriate cell density, the selected promoter is induced byappropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Yeast cells, e.g., Saccharomyces cerevisiae, employed in expression ofproteins can be manipulated as follows. Termite cellulase ortermite-associated symbiont cellulase polypeptides are generallysecreted by cells and therefore can be easily recovered from supernatantusing methods known to those of skill in the art. Proteins can also berecovered and purified from recombinant yeast cell cultures by methodsincluding spheroplast preparation and lysis, cell disruption using glassbeads, and cell disruption using liquid nitrogen for example.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences.

Additional methods include ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography, gel filtration,and lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

The host cells of the present invention can express cellulases in asecreted and/or a tethered form. For example, in some embodiments, thetermite cellulase or termite-associated symbiont cellulase polypeptideof the present invention can be in a secreted or a tethered form. Asused herein, a protein is “tethered” to an organism's cell surface if atleast one terminus of the protein is bound, covalently and/orelectrostatically for example, to the cell membrane or cell wall. Itwill be appreciated that a tethered protein may include one or moreenzymatic regions that may be joined to one or more other types ofregions at the nucleic acid and/or protein levels (e.g., a promoter, aterminator, an anchoring domain, a linker, a signaling region, etc.).While the one or more enzymatic regions may not be directly bound to thecell membrane or cell wall (e.g., such as when binding occurs via ananchoring domain), the protein is nonetheless considered a “tetheredenzyme” according to the present specification.

Tethering can, for example, be accomplished by incorporation of ananchoring domain into a recombinant protein that is heterologouslyexpressed by a cell, or by prenylation, fatty acyl linkage, glycosylphosphatidyl inositol anchors or other suitable molecular anchors whichmay anchor the tethered protein to the cell membrane or cell wall of thehost cell. A tethered protein can be tethered at its amino terminal endor optionally at its carboxy terminal end.

As used herein, “secreted” means released into the extracellular milieu,for example into the media. Although tethered proteins may havesecretion signals as part of their immature amino acid sequence, theyare maintained as attached to the cell surface, and do not fall withinthe scope of secreted proteins as used herein.

The termite cellulase or termite-associated symbiont cellulasepolypeptides of the present invention may be in the form of the secretedprotein, including the mature form, or may be a part of a largerprotein, such as a fusion protein. It is often advantageous to includean additional amino acid sequence which contains secretory or leadersequences, pro-sequences, sequences which aid in purification, such asmultiple histidine residues, or an additional sequence for stabilityduring recombinant production.

Secretion of desired proteins into the growth media has the advantagesof simplified and less costly purification procedures. It is well knownin the art that secretion signal sequences are often useful infacilitating the active transport of expressible proteins across cellmembranes. The creation of a transformed host capable of secretion maybe accomplished by the incorporation of a DNA sequence that codes for asecretion signal which is functional in the host production host.Methods for choosing appropriate signal sequences are well known in theart (see for example EP 546049; WO 9324631). The secretion signal DNA orfacilitator may be located between the expression-controlling DNA andthe instant gene or gene fragment, and in the same reading frame withthe latter.

The host cells of the present invention can express one or more termiteor termite-associated symbiont cellulase polypeptides. The host cells ofthe present invention can also express, in addition to the termite ortermite-associated symbiont cellulase, cellulases from other organisms.For example, the host cells of the present invention can express, inaddition to the termite or termite-associated symbiont cellulase theSchizochytrium aggregatum Cbh1 protein. In some embodiments, the hostcell expresses at least one endogluconase, at least one exogluconase andat least one β-glucosidase, wherein at least one of the endogluconase,exogluconase or β-glucosidase is a termite or termite-associatedsymbiont cellulase. In some embodiments, the host cell expresses atleast two endogluconases, at least two exogluconases, or at least twoβ-glucosidases. In some embodiments, the host cell expresses at leastone cellulase that has both endogluconase and exogluconase activity andat least one additional cellulase that has β-glucosidase activity.

The transformed host cells or cell cultures, as described above, can beexamined for endoglucanase, cellobiohydrolase and/or β-glucosidaseprotein content. Protein content can be determined by analyzing the host(e.g., yeast) cell supernatants. In certain embodiments, the highmolecular weight material is recovered from the yeast cell supernatanteither by acetone precipitation or by buffering the samples withdisposable de-salting cartridges. The analysis methods include thetraditional Lowry method or protein assay method according to BioRad'smanufacturer's protocol. Using these methods, the protein content ofsaccharolytic enzymes can be estimated.

The transformed host cells or cell cultures, as described above, can befurther analyzed for hydrolysis of cellulose (e.g., by a sugar detectionassay), for a particular type of cellulase activity (e.g., by measuringthe individual endoglucanase, cellobiohydrolase or β-glucosidaseactivity) or for total cellulase activity. Endoglucanase activity can bedetermined, for example, by measuring an increase of reducing ends in anendogluconase specific CMC substrate. Cellobiohydrolase activity can bemeasured, for example, by using insoluble cellulosic substrates such asthe amorphous substrate phosphoric acid swollen cellulose (PASC) ormicrocrystalline cellulose (Avicel) and determining the extent of thesubstrate's hydrolysis. β-glucosidase activity can be measured by avariety of assays, e.g., using cellobiose.

A total cellulase activity, which includes the activity ofendoglucanase, cellobiohydrolase and β-glucosidase, will hydrolyzecrystalline cellulose synergistically. Total cellulase activity can thusbe measured using insoluble substrates including pure cellulosicsubstrates such as Whatman No. 1 filter paper, cotton linter,microcrystalline cellulose, bacterial cellulose, algal cellulose, andcellulose-containing substrates such as dyed cellulose, alpha-celluloseor pretreated lignocellulose.

One aspect of the invention is thus related to the efficient productionof cellulases, especially termite and termite-associated symbiontcellulases, to aid in the digestion of cellulose and generation ofethanol. A cellulase can be any enzyme involved in cellulase digestion,metabolism and/or hydrolysis, including an endogluconase, exogluconase,or β-glucosidase.

It will be appreciated that suitable lignocellulosic material may be anyfeedstock that contains soluble and/or insoluble cellulose, where theinsoluble cellulose may be in a crystalline or non-crystalline form. Invarious embodiments, the lignocellulosic biomass comprises, for example,wood, corn, corn cobs, corn stover, corn fiber, sawdust, bark, leaves,agricultural and forestry residues, grasses such as switchgrass, cordgrass, rye grass or reed canary grass, miscanthus, ruminant digestionproducts, municipal wastes, paper mill effluent, newspaper, cardboard,miscanthus, sugar-processing residues, sugarcane bagasse, agriculturalwastes, rice straw, rice hulls, barley straw, cereal straw, wheat straw,canola straw, oat straw, oat hulls, stover, soybean stover, forestrywastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood,softwood or combinations thereof.

In certain embodiments of the present invention, a host cell comprisinga vector which encodes and expresses a termite cellulase ortermite-associated symbiont cellulase that is utilized for consolidatedbioprocessing is co-cultured with additional host cells expressing oneor more additional endoglucanases, cellobiohydrolases and/orβ-glucosidases. In other embodiments of the invention, a host celltransformed with a termite cellulase or termite-associated symbiontcellulase is transformed with and/or expresses one or more otherheterologous endoglucanases, exogluconases or β-glucosidases. Theendogluconase, exogluconase and/or β-glucosidase can be any suitableendogluconase, exogluconase and β-glucosidase derived from, for example,a termite, fungal or bacterial source.

Specific activity of cellulases can also be detected by methods known toone of ordinary skill in the art, such as by the Avicel assay (describedsupra) that would be normalized by protein (cellulase) concentrationmeasured for the sample. To accurately measure protein concentration atermite or termite-associated symbiont cellulase can be expressed with atag, for example a His-tag or HA-tag and purified by standard methodsusing, for example, antibodies against the tag, a standard nickel resinpurification technique or similar approach.

In additional embodiments, the transformed host cells or cell culturesare assayed for ethanol production. Ethanol production can be measuredby techniques known to one or ordinary skill in the art e.g. by astandard HPLC refractive index method.

EXAMPLES Materials and Methods Media and Strain Cultivation

TOP10 cells (Invitrogen) were used for plasmid transformation andpropagation. Cells were grown in LB medium (5 g/L yeast extract, 5 g/LNaCl, 10 g/L tryptone) supplemented with ampicillin (100 mg/L).Also, 15g/L agar was added when solid media was desired.

Yeast strains were routinely grown in YPD (10 g/L yeast extract, 20 g/Lpeptone, 20 g/L glucose), or YNB+glucose (6.7 g/L Yeast Nitrogen Basewithout amino acids, and supplemented with appropriate amino acids forstrain, 20 g/L glucose). 15 g/L agar was added for solid media.

Yeast strain MO375 was used as a host strain in several experiments.MO375 was derived from Y294 (MO013) in which His3 and Trp1 auxotrophieswere rescued by transformation with S. cerevisiae His3 and Trp1 PCRproducts. Y294 (ATCC 201160) has the following genotype: a leu2-3,112ura3-52 his3 trp1-289.

Molecular Methods

Standard protocols were followed for DNA manipulations (Sambrook et al.1989). PCR was performed using Phusion polymerase (New England Biolabs)for cloning, and Taq polymerase (New England Biolabs) for screeningtransformants, and in some cases Advantage Polymerase (Clontech) for PCRof genes for correcting auxotrophies. Manufacturers guidelines werefollowed as supplied. Restriction enzymes were purchased from NewEngland Biolabs and digests were set up according to the suppliedguidelines. Ligations were performed using the Quick ligation kit (NewEngland Biolabs) as specified by the manufacturer. Gel purification wasperformed using either Qiagen or Zymo research kits, PCR product anddigest purifications were performed using Zymo research kits, and Qiagenmidi and miniprep kits were used for purification of plasmid DNA.

Yeast Transformation

Yeast were transformed using LiOAc chemical transformation.Specifically, yeast were grown in 2 mls of YPD at 30° C. overnight. Thefollowing morning, 50 mls of YPD were inoculated with 0.5 mls of theovernight culture and then grown at 30° C. with shaking for 4-5 hours.Cells were then spun down at top speed in a clinical centrifuge forabout 5 minutes. The supernatant was removed and the cells wereresuspended in water and spun down again. Next, the cells wereresuspended in 1 ml of 100 mM LiOAc and transferred to a microfuge tube.Cells were spun at top speed for 15 seconds and then suspended in 150 μltransformation mix (15 μl H₂O, 15 μl 1 M LiOAc, 20 μl DNA carrier(Ambion catalog number AM9680) and 100 μl 50% PEG 3350). Miniprep DNA (1μl) and 150 μl of the transformation mix containing yeast cells weremixed in a microfuge tube, incubated at 30° C. for 30 minutes and thenheatshocked for 15 minutes in a 42° C. water bath. After the heatshock,cells were spun down for 15 seconds, the transformation mix was removedby pipette, and 50 μl of sterile water was added. Cells were gentlyresuspended and plated on selective media and grown for 2-3 days at 30°C.

Alternatively, yeast were transformed by electrotransformation. Aprotocol for electrotransformation of yeast was developed based on Cho KM et al., “Delta-integration of endo/exo-glucanase and beta-glucosidasegenes into the yeast chromosomes for direct conversion of cellulose toethanol,” Enzyme Microb Technol 25:23-30 (1999) and on Ausubel et al.,Current protocols in molecular biology. USA: John Wiley and Sons, Inc.(1994). Yeast cells for transformation were prepared by growing tosaturation in 5 mL YPD cultures. 4 mL of the culture was sampled, washed2× with cold distilled water, and resuspended in 640 μL cold distilledwater. 80 μL of 100 mM Tris-HCl, 10 mM EDTA, pH 7.5 (10×TE buffer—filtersterilized) and 80 μL of 1M lithium acetate, pH 7.5 (10× liAc—filtersterilized) were added and the cell suspension was incubated at 30° C.for 45 min. with gentle shaking. 20 μL of 1M DTT was added andincubation continued for 15 min. The cells were then centrifuged, washedonce with cold distilled water, and once with electroporation buffer (1Msorbitol, 20 mM HEPES), and finally resuspended in 267 μLelectroporation buffer.

For electroporation, 100 ng of plasmid DNA (pRDH105) was combined with˜100 ng of His3 PCR product and added to 50 μL of the cell suspension ina sterile 1.5 mL microcentrifuge tube. A control strain was built byusing 100 ng each of the Ura3 and His 3 PCR products. The mixture wasthen transferred to a 0.2 cm electroporation cuvette, and a pulse of 1.4kV (200Ω, 25 μF) was applied to the sample using the Biorad Gene Pulserdevice. 1 mL of cold 1M sorbitol adjusted to was placed in the cuvetteand the cells were spread on Yeast nitrogen base media (Difco) withglucose, and not supplemented with amino acids.

Cellulase Assay Reagents

Avicel substrate mix was prepared by combining 0.6 g Avicel (2%), 500 μl3 M sodium acetate pH 5.0 (50 mM), 1.2 ml 0.5% sodium azide (0.02%) and30 μl BGL (Novozyme-188, Sigma) and adding dH₂0 to a total volume of 30mls.

Carboxymethylcellulose (CMC) mix was prepared by a mixing 1.14 g CMC per100 mL citrate buffer (50 mM pH 5.5) and autoclaving for 20-25 minutes.The CMC/citrate buffer mixture was agitated to ensure that all CMC wasdissolved. 1 ml of 0.5% of sodium azide was added to the 44 mls ofCMC/citrate buffer mixture to prepare 45 mls of the final CMC mix.

DNS 1% was prepared by mixing 10 g 3,5-dinitrosalicylic acid, 0.5 gsodium sulfite, 10 g sodium hydroxide and water to 1 liter. DNS wascalibrated with glucose, using glucose samples with concentration of 0,1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 g/L, and the slope (S) was calculatedsuch that S=0.1 at 565 nm.

Calculations of the percent Avicel or CMC converted after about 24and/or 48 hours were performed using the following equation:

$Y = {\frac{\left( {{{OD}\left( {T = {24\mspace{14mu} {or}\mspace{14mu} 48}} \right)} - {{OD}\left( {T = 0} \right)}} \right) \times 100\%}{S \times A} = {\frac{\Delta \; {OD} \times 100}{0.1 \times 10} = {\Delta \; {OD} \times 100}}}$

wherein Y=% of Avicel or CMC converted at 24 or 48 hrs; S=DNS/glucosecalibration slope at 565 nm; and A=Avicel or CMC concentration at T=0.

Example 1 Cloning of Codon-Optimized Termite Cellulase Genes and theirExpression in Saccharomyces cerevisiae

Cellulase genes from various termite sources (as indicated in Table 5below) were codon-optimized for expression in the yeast Saccharomycescerevisiae and K. lactis.

TABLE 5 Termite and Termite-Associated Cellulase Symbiont CellulaseConstructs and Strains Yeast Expression strain construct Family OrganismNCBI ref Activity M0443 pMU456 Protozoa Holomastigotoides mirabileAB071011 endo M0444 pMU457 Protozoa Reticulitermes speratus symbiontAB274534 endo M0446 pMU465 Protozoa Coptotermes lacteus symbiontAB089801 endo M0447 pMU466 Protozoa Reticulitermes speratus symbiontAB045179 endo M0449 pMU471 Metazoa Coptotermes formosanus AB058671 endoM0450 pMU472 Metazoa Nasutitermes takasagoensis AB013272 endo M0451pMU473 Metazoa Coptotermes acinaciformis AF336120 endo M0452 pMU490Protozoa Cryptocercus punctulatus symbiont AB274702 endo M0453 pMU491Protozoa Mastotermes darwiniensis symbiont AB274656 endo M0454 pMU492Protozoa Pseudotrichonympha grassii AB071864 exo M0455 pMU493 ProtozoaReticulitermes flavipes gut symbiont DQ014511 endo M0460 pMU499 MetazoaMastotermes darwinensis AJ511343 endo M0461 pMU500 MetazoaReticulitermes speratus AB019095 endo M0462 pMU501 ProtozoaHodotermopsis sjoestedti symbiont AB274582 endo M0463 pMU502 MetazoaReticulitermes flavipes AY572862 endo M0464 pMU503 Metazoa Nasutitermeswalkeri AB013273 endo M0465 pMU504 Metazoa Panesthia cribrata AF220597endo M0480 pMU468 Protozoa Neotermes koshunensis symbiont AB274614 endo

For metazoan genes, the native signal sequence was replaced with S.cerevisiae alpha mating factor pre signal sequence with the followingamino acid sequence: MRFPSIFTAVLFAASSALA (SEQ ID NO: 43). For protozoangenes, native signal sequences could not be detected; therefore, the S.cerevisiae alpha mating factor pre signal sequence was attached to the5′ end of the gene. When necessary to optimize the protein sequenceafter signal peptidase cleavage, codons encoding several N-terminalamino acids of the cellulase were removed.

The codon optimized sequences used in the following experiments areshown in Table 3 above. The synthetic sequences were then cloned intothe episomal yeast expression vector (pMU451) under control of ENO1promoter and terminator into PacI/AscI sites (see FIG. 1), and theresulting expression constructs are listed in Table 5

These constructs were then utilized to transform S. cerevisiae strainMO375 host strain. The resulting yeast strains, which are listed inTable 5, were tested for cellulase activity according to the proceduresdescribed in the following examples.

Example 2 Avicel Conversion Assay

An Avicel conversion assay was used to determine the activity of S.cerevisiae containing termite cellulases and termite-associatedsymbiotic protist cellulases. In these experiments, the strains to betested were inoculated in 600 μl of YPD in a deep 96-well plate andgrown with shaking at 30° C. for three days. Then, cells were spun atmaximum speed for 10 minutes. Avicel substrate mix (300 μl) was added towells of a new deep 96-well plate, and shaking was repeated throughoutaddition to prevent Avicel from settling. Then 300 μl of yeastsupernatant (or buffer for negative control) was added to the wellscontaining the Avicel substrate. The yeast supernatant and substratewere mixed by pipetting and then 100 μl was transferred to a 96-well PCRplate for a sample at T=0. The deep 96-well plate containing yeastsupernatant and substrate was incubated at 35° C. with shaking at 800rpm. The 96-well PCR plate containing the T=0 samples was spun at 2000rpm for 2 minutes. The supernatant (50 μl) was transferred to a new96-well PCR plate that contained 100 μl of DNS mix in each well. The PCRplate containing the supernatant and DNS mix was heated at 99° C. forfive minutes and then cooled to 4° C. in a PCR machine. After cooling to4° C., 50 μl was transferred to a micro titer plate and the absorbanceat 565 nm was measured using a plate reader. Samples were removed fromthe deep 96-well plate containing yeast supernatant and substrate thatwas incubated at 35° C. with shaking at 800 rpm at approximately 24 and48 hours and the samples were processed to determine absorbanceaccording to the same procedures as described for the samples obtainedat T=0.

Each strain was tested four times, and the % Avicel conversion wascalculated. Strain MO423, containing T. reesei EG1 was used as apositive control. Strain MO419, which was created by transforming MO375with empty pMU451 vector, was assayed as a negative control. As shown inFIG. 2, many of the cellulases tested demonstrated activity on Avicel.

Example 3 Carboxymethyl-Cellulose Conversion Assay

A Congo Red carboxymethyl-cellulose (CMC) assay was used to test theactivity of S. cerevisiae containing termite cellulase ortermite-associated symbiotic protist genes. In these experiments, yeastcolonies were patched on yeast nitrogen base (YNB) plates with CMC.(Plates were made by mixing 0.5 g CMC, 10 g Agar, 10 g glucose and waterto 450 mls, autoclaving the mixture, and then adding 50 mls YNB withamino acids.) Plates were grown for two days at 30° C. and then washedwith 1 M tris pH 7.5. Colonies were then stained for 20 minutes in CongoRed (0.1% in H₂O) and washed several times with 1 M NaCl. The photographof FIG. 4 was taken shortly after destaining to avoid increases inbackground over timer. Strain MO423, containing T. reesei EG1 was usedas a positive control, and MO247, a strain expressing T. emersonii CBH1in a vector similar to pMU451 with furl gene knocked-out to make theepisomal plasmid stable, was used as a reference of activity of anexogluconase in the Congo Red assay.

Cellulase activity on CMC was then quantitated in several of the strainsusing a CMC conversion assay. In this assay, yeast strains to be testedwere inoculated in 10 mls media in 50 ml tubes and grown with shakingfor 3 days. Tubes were then spun at max speed for 10 minutes to obtainyeast supernatant. Assays were performed in 96-well plates, and fourreplicates were performed for each strain tested.

Yeast supernatant (50 μl) (or buffer for negative control), was added towells of a deep well 96-well plate containing CMC mix (450 μl) and mixedby pipetting. A 50 μl aliquot was then removed and transferred to a wellof a 90-well PCR plate containing 100 μl DNS 1%. The deep well 96-wellplate was incubated at 35° C. at 800 rpm for approximately 24 hours. ThePCR plate was heated to 99° C. for 5 minutes and then cooled to 4° C. ina PCR machine. The 50 μl samples in the PCR plates were transferred to amicrotiter plate and the absorbance of each sample was read at 565 nm.After the deep well 96-well plates had incubated for 24 hours, sampleswere transferred to a plate containing DNS 1%, heated and cooled in aPCR machine and transferred to a microtiter plate for absorbance readingas described. The percentage of CMC converted was calculated for allsamples. As in the Avicel assay, MO419 was used as an empty vector,negative control. The results shown in FIG. 4 demonstrate that each ofthe strains tested showed increased activity as compared to yeastexpressing T. reesei EG1 (positive control).

In addition, strain MO446 was tested in the CMC assay (data not shown)and did not show any activity on CMC. MO446 expresses a protistCoptotermes lacteus symbiont cellulase (gene accession #AB089801) thathas been annotated as an endo-beta-1,4,gluconase. However, the lack ofactivity on CMC, in addition to the significant activity on Avicel(demonstrated in Example 2 and FIG. 2) indicate that AB089801 may infact be an exogluconase.

Example 4 Yeast-Made Termite Endoglucanase Significantly IncreasesAvicel Conversion by Yeast-Made Fungal CBHs

To determine if the addition of termite endoglucanase to fungal CBHs hasa positive effect on Avicel conversion, an Avicel assay was performedwith a yeast-made fungal CBH mix (Talaromyces emersonii CBH1+CBD (T.reesei CBH1) and Chrysosporium lucknowense CBH2b), as well as the fungalCBH mix combined with yeast-made Coptotermes formosanus endoglucanase(CfEG) (FIG. 4).

Table 6 below describes the samples that were used in this experiment:

TABLE 1 Enzymes used in experiment with termite EG added to fungal CBHsProduction Expression Sample Protein Enzyme strain vector preparationmg/l Talaromyces M0759 pMU624 (2u) 1 l fermenter 290 emersonii CBH1 + T.reesei CBH1 CBD (TeCBH1 + CBD) Chrysosporium M0969 pMU784 (2u) 1 lfermenter 800 lucknowense CBH2b (ClCBH2b) Coptotermes M0968 pMU471 (2u)100 ml  90 formosanus pMU663 shake flask endoglucanase (delta) (CfEG)Negative control M0509 none 1 l fermenter n/a

All strains used are derivatives from the industrial S. cerevisiaestrain MO509. TrCBH1+CBD and ClCBH2b are expressed from episomal 2μvectors (pMU624 and pMU784) with a pMU451 backbone that has beendescribed above. In the CfEG-producing M0968 strain, endoglucanase (EG)was first introduced on the 2μ vector pMU471 (pMU451 backbone). Later,the CfEG copy number was increased by transformation with the deltaintegration expression vector pMU663 (pMU562 backbone). In allexpression vectors, the coding gene was inserted into PacI/AscI sites ofthe pMU451 or pMU562 backbones between the ENO1 promoter and terminator.Strains M0759, M0969 and M0509 were grown in 1 liter (L) fermenters inYPD-based media plus 50 g/L glucose with 24 hrs batch cultivationfollowed by a stepped feed of 50% glucose with vitamins and traceelements for another 24 hrs. M0968 was grown in 100 milliliters (ml) YPDin a 500 ml shake flask for 3 days. Supernatants of the strains wereused in the assay as an enzyme source. Protein concentration wasmeasured by HPLC. To make the CBH mix for the Avicel assay, CBH1 andCBH2 samples were mixed in a 4:1 ratio. For the negative control (NegCont) 300 μl of the M0509 strain supernatant was used in the assay. Inthe “CBH” sample, 200 μl of the M0509 supernatant was added to 100 μl ofthe CBH mix. In the “CBH+EG” sample, 200 μl of the M0968 supernatant wasadded to 100 μl of the CBH mix.

FIG. 5 shows that the addition of yeast-made CfEG significantlyincreased Avicel conversion by yeast-made fungal CBHs. Yeast-madeendoglucanase may be synergistically acting with yeast-made fungal CBHs.The CfEG sample alone will be tested to quantitatively evaluate andconfirm this synergistic effect. The results discussed above indicatethat the cellulytic properties of yeast-expressing fungal CBHs can besignificantly improved by integration of a termite CfEG.

Example 5 Ethanol Production from Avicel by Co-Culture of CellulyticYeast Strains Including Strain Producing Termite Endoglucanase (CfEG)

To evaluate the improved performance of yeast strains each expressing adifferent cellulase (TeCBH1+CBD, ClCBH2b, CfEG, and Saccharomycopsisfibuligera BGL1 (SfBGL)), ethanol production from 10% Avicel wasmeasured in a SSF shake flask.

Each of the strains referred to above has a MO013 background (the Y294yeast strain: genotype: a leu2-3,112 ura3-52 his3 trp1-289; ATCC No.201160) with cellulases expressed on an episomal 2μ plasmid (pMU451backbone with coding gene inserted into PacI/AscI sites). The Furl genewas knocked out in these strains to stabilize the plasmid. Four strainseach expressing one cellulase (M0595−TeCBH1+CBD; M0563−ClCBH2;M0592−CfEG; M0566−SfBGL1) were pre-grown separately in YPD in shakeflasks for 3 days, mixed in equal proportion, and transferred (10%inoculation volume) into several nitrogen purged pressure bottles withYP+10% of Avicel and different concentrations of external cellulases(Zoomerase, Novozyme). The total volume was 30 ml. The bottles wereincubated at 35° C. with shaking for 160 hrs and the samples were takenduring this time for ethanol concentration measurement (by HPLC). Theexperiment was also performed with the control non-cellulytic MO249strain (FIG. 6). FIG. 6 clearly demonstrates that co-culture ofcellulytic yeast strains performs significantly better compared to theparental non-cellulytic strain at all concentrations of external enzymesused due to the efficient contribution of endogenously producedcellulases.

The above-described results demonstrate that the four cellulases(TeCBH1+CBD, ClCBH2b, CfEG, and Saccharomycopsis fibuligera BGL1(SfBGL)) can be functionally expressed in yeast. In combination, thesefour cellulases provide a significant level of cellulase activity. Infact, a two-fold less amount of enzyme is required (as compared to theempty control strain) when a co-culture of cells expressing these fourcellulases is utilized, to achieve the same amount of ethanolproduction. Thus, the co-culturing of these particular cellulaseexpressing cells, including the expression of termite EG, significantlyimproves ethanol production from Avicel.

In order to make the contribution of yeast-made enzymes even morequantitative, a theoretical ethanol yield at 160 hrs of SSF was plottedagainst external cellulase loads (FIG. 7). FIG. 7 demonstrates thatco-cultured cellulytic yeast strains save more than 50% of externalenzymes. This demonstrates the feasibility of a yeast-based CBP concept.

Example 6 Quantitative Analysis of Termite Endoglucanase and OtherCellulases Produced by Yeast During Fermentation

To investigate the ability of yeast to produce and accumulate cellulasesduring high cell density fermentation, the strain M0712 expressing fourcellulases (SfBGL, CfEG, ClCBH2b, and TeCBH1+CBD) was cultivated in a 3Lbioreactor.

The M0712 strain is a derivative of the robust M0509 where all fourcellulases are expressed from delta integration constructs with thezeocin marker (coding cellulase genes inserted into PacI/AscI sites ofpMU562 backbone). YPD-based rich media with additional vitamins, traceelements, and 6.7 g/L of yeast nitrogen base was used for batch culturewith 50 g/L glucose. After cultivation in batch phase for 24 hours, astepped feed of 50% glucose with vitamins and trace elements was carriedout for another 36 hours. At several time points, reactor samples weretaken and the dry cell weight was measured. Additionally, proteinconcentration for each cellulase was measured by HPLC (FIG. 8). A celldensity of ˜90 g/L DCW was achieved in the run, as well as a totalcellulase concentration of ˜1.4 g/L (not including SfBGLI expression).

FIG. 8 demonstrates that yeast were able to accumulate termiteendoglucanase to about 900 mg/l or 10 mg/g cells. This data means thatyeast were able to produce CfEG up to 2% of TCP (total cell protein)which is a significant level of heterologous protein production in S.cerevisiae.

Example 7 Synergy Between EGs and CBH1

An Avicel assay was performed (as described in Example 4) using T.emersonii CBH1 (with no CBD) mixed with different termite endoglucanases(EGs) as well as with T. reesei EG1. The yeast strains utilized in thisexperiment were created by expressing the cellulases from the pMU451vector (described above) in an M0375 background strain. All yeaststrains were grown in 10 ml YPD in 50 ml conical tubes for 3 days at 30°C. and 250 rpm. The Avicel assay was performed using supernatants fromthe different strains, both singly and combined. For single strains, 300μl of supernatant was used; for the combined samples, 150 μl of eachstrain was mixed together for the assay (See FIG. 9).

FIG. 9 demonstrates that combination of Coptotermes formosanus EG (CfEG)with T. emersonii CBH1 (TeCBH1) provides the highest Avicel conversion.Moreover, there is synergy between TeCBH1 and CfEG, with the degree ofsynergy about 2 at both the 24 and 48 hour time points. The degree ofsynergy was calculated as the synergistic activity on Avicel of theTeCBH1/CfEG mix divided by the sum of activities of the individualcomponents (after the negative control value was deducted). In thecombination experiments, the two-fold dilution of the single enzymesamples was also factored into the calculations.

These examples illustrate possible embodiments of the present invention.While the invention has been particularly shown and described withreference to some embodiments thereof, it will be understood by thoseskilled in the art that they have been presented by way of example only,and not limitation, and various changes in form and details can be madetherein without departing from the spirit and scope of the invention.Thus, the breadth and scope of the present invention should not belimited by any of the above-described exemplary embodiments, but shouldbe defined only in accordance with the following claims and theirequivalents.

All documents cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedor foreign patents, or any other documents, are each entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited documents.

1. An isolated polynucleotide comprising a nucleic acid fragment whichencodes at least 50 contiguous amino acids of a cellulase, wherein saidnucleic acid fragment is codon-optimized for expression in a yeaststrain and wherein the cellulase is a termite cellulase or atermite-associated symbiont cellulase.
 2. (canceled)
 3. (canceled) 4.The polynucleotide of claim 1, wherein the yeast is selected from thegroup consisting of Saccharomyces cerevisiae, Kluveromyces lactus,Kluyveromyces marxianus, Schizzosaccharomyces pombe, Candida albicans,Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenulapolymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans,Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomycespombe and Schwanniomyces occidentalis.
 5. (canceled)
 6. Thepolynucleotide of claim 1, wherein the cellulase has exogluconaseactivity.
 7. The polynucleotide of claim 1, wherein the cellulase hasendogluconase activity.
 8. (canceled)
 9. The polynucleotide of claim 1,wherein the cellulase has β-glucosidase activity.
 10. The polynucleotideof claim 1, wherein the cellulase is a protozoan cellulase.
 11. Thepolynucleotide of claim 10, wherein the cellulase is a Holomastigotoidesmirabile, Reticulitermes speratus symbiont, Coptotermes lacteussymbiont, Reticulitermes speratus symbiont, Cryptocercus punctulatussymbiont, Mastotermes darwiniensis symbiont, Pseudotrichonympha grassii,Reticulitermes flavipes gut symbiont, Hodotermopsis sjoestedti symbiontor Neotermes koshunensis symbiont cellulase.
 12. The polynucleotide ofclaim 1, wherein the cellulase is a metazoan cellulase.
 13. Thepolynucleotide of claim 12, wherein the cellulase is a Coptotermesformosanus, Nasutitermes takasagoensis, Coptotermes acinaciformis,Mastotermes darwinensis, Reticulitermes speratus, Reticulitermesflavipes, Nasutitermes walkeri or Panesthia cribrata cellulase.
 14. Thepolynucleotide of claim 1, wherein the cellulase is a bacterialcellulase.
 15. The polynucleotide of claim 1, wherein the cellulase is afungal cellulase.
 16. The polynucleotide of claim 1, wherein thecellulase is a yeast cellulase and wherein said nucleic acid fragment iscodon-optimized for expression in a heterologous yeast strain. 17-20.(canceled)
 21. The polynucleotide of claim 1, wherein at least onenucleotide within a sequence of 4, 5, 6, 7, 8, 9, or 10 consecutive A,T, C or G nucleotides is replaced with a different nucleotide, whereinthe nucleotide replacement does not alter the amino acid sequenceencoded by the polynucleotide and wherein the nucleotide replacementcreates a codon that is the second most frequently used codon to encodean amino acid in Saccharomyces cerevisiae.
 22. The polynucleotide ofclaim 1, wherein at least one restriction enzyme site within thepolynucleotide is removed by replacing at least one nucleotide withinthe restriction enzyme site with a different nucleotide, wherein thenucleotide replacement does not alter the amino acid sequence encoded bythe polynucleotide and wherein the nucleotide replacement creates acodon that is the second most frequently used codon to encode an aminoacid in Saccharomyces cerevisiae.
 23. (canceled)
 24. The polynucleotideof claim 1, wherein one or more direct repeats, inverted repeats andmirror repeats with lengths of 10 bases or longer within saidpolynucleotide is altered by replacing at least one nucleotide withinthe repeat with a different nucleotide, wherein the nucleotidereplacement does not alter the amino acid sequence encoded by thepolynucleotide and wherein the nucleotide replacement creates a codonthat is the second most frequently used codon to encode an amino acid inSaccharomyces cerevisiae.
 25. The polynucleotide of claim 1, whereinsaid polynucleotide is operably associated with a heterologous nucleicacid. 26-31. (canceled)
 32. The polynucleotide of claim 1, wherein thenucleic acid fragment comprises a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 1-20.
 33. A vector comprising afirst polynucleotide, wherein said first polynucleotide is thepolynucleotide of claim
 1. 34-53. (canceled)
 54. A host cell comprisinga polynucleotide encoding at least 50 contiguous amino acids of aheterologous cellulase, wherein the heterologous cellulase is a termitecellulase or a termite-associated symbiont cellulase, wherein the hostcell is a yeast cell and wherein the heterologous cellulase isexpressed. 55-86. (canceled)
 87. A host cell comprising thepolynucleotide of claim
 1. 88-90. (canceled)
 91. The host cell of claim54, wherein the host cell has the ability to saccharify crystallinecellulose.
 92. (canceled)
 93. A method for hydrolyzing a cellulosicsubstrate, comprising contacting said cellulosic substrate with a hostcell according to claim
 54. 94. (canceled)
 95. A method of fermentingcellulose using the host cell of claim 54, said method comprisingculturing said host cell in medium that contains crystalline celluloseunder suitable conditions for a period sufficient to allowsaccharification and fermentation of the cellulose.
 96. The method ofclaim 95, wherein said host cell produces ethanol.
 97. Thepolynucleotide of claim 1, wherein said polynucleotide encodes aCoptotermes formosanus endoglucanase (CfEG).
 98. The host cell of claim54, wherein said host cell expresses CfEG.
 99. The host cell of claim 54co-cultured with one or more additional host cells, wherein each of theone or more additional host cells expresses one or more endoglucanases,cellobiohydrolases and/or β-glucosidases.
 100. (canceled)
 101. A methodfor hydrolyzing a cellulosic substrate, comprising contacting saidcellulosic substrate with a host cell according to claim 98.